WO1997015670A1 - Novel subtilisin inhibitors - Google Patents

Abstract

The invention relates to novel subtilisin inhibitors comprising a human standard mechanism inhibitor that has the reactive site of inhibition replaced with the reactive site from a known subtilisin inhibitor. The invention provides nucleic acids, proteins, and methods for inhibiting subtilisins, and detergents containing modified subtilisin inhibitors.

Description

NOVEL SUBTILISIN INHIBITORS

FIELD OF THE INVENTION

The invention relates to novel subtilisin inhibitors comprising a human standard mechanism inhibitor that has the reactive site of inhibition replaced with the reactive site from a known subtilisin inhibitor. The invention provides nucleic acids, proteins, and methods for inhibiting subtilisins, and detergents containing modified subtilisin inhibitors.

BACKGROUND OF THE INVENTION

Several serine proteases have use in commercial processes. For example, subtilisin, a serine protease from Bacillus species, is used extensively in laundry detergent formulations. Detergent formulations containing enzymes, and particularly subtilisin, may exhibit enzyme stability problems upon storage, especially liquid detergents which can contain high levels of surfactants and water. An enzyme may hydrolyze in water and may degrade itself or other enzymes, resulting in the addition of extra enzyme or other costly additives to retard degradation.

Thus a variety of attempts have been made to stabilize detergent formulations to avoid the loss of enzymatic activity. See for example U.S. Patent Nos. 5,039,446, and 5,178,789, and cited documents, and European Patent Nos. 0 199 405 Bl and 0 549 675 Bl.

In addition, the allergenicity ofthe enzyme is also problematic for workers involved in producing the enzyme and the detergents containing them. In the case of subtilisin, particular allergenicity is associated with the active enzyme, which may cause proteolytic degradation of other proteins, including host proteins, leading to an immune response. Thus many techniques to limit exposure of workers to the active enzyme have been explored, including granularization, immobilization and inhibition ofthe enzyme, in an attempt to avoid the release of proteinaceous material during normal handling and storage.

However, the added inhibitor may itself be allergenic, such as the case with the ovomucoid third domain inhibitors, which are potent inhibitors of subtilisin.

Therefore, it is desirable to make a subtilisin protease inhibitor which does not itself cause a significant allergic reaction. WO 92/10755 describes methods for selecting the immunological epitopes of a protein for the purpose of changing the sequence and reducing the immunological response.

Accordingly, it is an object ofthe invention to provide subtilisin inhibitors which may be used to inhibit subtilisin to prevent or decrease an immune response to the inhibitor and the enzyme-inhibitor complex.

SUMMARY OF THE INVENTION

In accordance with the foregoing objects, it is an aspect ofthe invention to provide modified subtilisin inhibitors comprising a modified human standard mechanism inhibitor. At least one amino acid ofthe reactive site of a human standard mechanism inhibitor is substituted with a different amino acid, such that the K, ofthe modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor.

Also provided are modified subtilisin inhibitors wherein at least five amino acids ofthe reactive site ofthe human standard mechanism inhibitor are substituted with different amino acids.

Also provided are modified subtilisin inhibitors wherein the reactive site ofthe human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor.

Also provided are methods of inhibiting a target subtilisin comprising reversibly binding to a target subtilisin a modified subtilisin inhibitor. The modified subtilisin inhibitor comprises a human standard mechanism inhibitor wherein the reactive site ofthe human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor. The K, ofthe modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor.

Further provided are target subtilisins inhibited by a modified subtilisin inhibitor ofthe invention.

Additionally provided are nucleic acids encoding a modified human standard mechanism inhibitor, wherein at least one amino acid ofthe reactive site of said human standard mechanism inhibitor is substituted with a different amino acid, such that the K, ofthe modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor. Also provided are nucleic acids wherein the reactive site ofthe human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor.

Further provided are expression vectors and host cells comprising the nucleic acids ofthe invention.

Additionally provided are detergent compositions. The compositions comprise a modified subtilisin inhibitor ofthe invention and a detergent containing a target subtilisin.

BRIEF DESCRIPTION OF THE DRAWTNGS

Figure 1 depicts the pBN3 plasmid with the restriction sites shown, as described in Example 1.

Figure 2 depicts the pKKphoA.PSTI.SSI.M2 plasmid as described in Example

2.

Figure 3 depicts the standard mechansims inhibitors hPSTI, and acrosin inhibitor (depicted in the Figure as HUSI-II). Human fooistatin (FOLL), human agrin domain 7 (hAGR7) and human hevin (HEVIN) are also depicted; they all have sequence homology to hPSTI. They could also be used as backbones (scaffolds) for the modified protease inhibitors ofthe invention, although currently they have not been shown to inhibit proteases without additional modifications. The PIO to PIO' sites and the Pl-PT junctions are depicted (known for hPSTI and acrosin inhibitor; putative identification based on homology for the others). It should be noted for all ofthe sequences depicted in the Figures that the PIO to PIO' region is determined by homology, and thus the individual inhibitors may have a few more or a few less residues than the 20 residues ofthe PIO to PIO' site generally depicted. That is, the PIO to PIO' site is determined by homology to the hPSTI site depicted on the top line.

Figure 4 depicts the MPI-1, MPI-2 (domains 1 and 2 of mucous protease inhibitor), HE4-1, HE4-2 (domains 1 and 2 of human epididymis protease inhibitor) and SKALP inhibitors, with the P 10 to P 10' and P 1 -P 1 ' junctions depicted. As above, the PIO to PIO' site may have a few more or a few less residues.

Figure 5 depicts the ovomucoid third domains of 106 avian species, with the PIO to PIO' and Pl-Pl' junctions depicted. These sequences are originally reported in Laskowski et al., Biochem. 26:202-221 (1987), hereby expressly incoφorated by reference (additional sequences were submitted to the Protein Sequence Database; see 1990).

Figure 6 depicts the reactive sites ofthe SSI inhibitors, with the P10 to P10' and Pl-Pl' sites shown. As above, the P10 to P10' site is determined on the basis of homology to the top SSI sequence, and thus the actual PIO to PIO' site for some ofthe other inhibitors may have a few more or a few less residues in the lineup.

Figures 7 A and 7B depict the sequences ofthe red sea turtle inhibitor and rat agrin domain 5, with the P10 to P10' sites and the Pl-Pl' junctions shown based on homology to MPI-2 and hPSTI, respectively.

Figure 8 depicts the sequence ofthe hPSTI.M2 gene for baculoviral expression, with a BamHI site and a EcoRI site shown.

Figures 9A and 9B depict the sequence ofthe hPSTI.SSI inhibitor. Figure 9A depicts the hPSTI.SSI.M2 construction, for expression in E. coli using the phoA promoter, with restriction sites shown. The signal sequence is underlined, the reactive site from SSI is shown in bold, and the M2 tag is in italics. The first alanine after the signal sequence is from the cloning vector, and is not necessary. Figure 9B depicts the amino acid sequence ofthe hPSTI.SSI modified subtilisin inhibitor without the M2 tag or the N-terminal alanine.

Figures 10A and 10B depict the sequence ofthe hPSTI.OMTKY3 modified subtilisin inhibitor. Figure 10A the sequence ofthe hPSTI.OMTK-Y3.M2 construction, with restriction sites shown. The signal sequence is underlined, the reactive site from SSI is shown in bold, and the M2 tag is in italics. Figure 10B depicts the amino acid sequence ofthe hPSTI.OMTKY3 modified subtilisin inhibitor without the M2 tag or the N-terminal alanine.

Figures 11 A and 1 IB depict the sequence ofthe hPSTI.SSImutant modified subtilisin inhibitor. Figure 11 A depicts the sequence ofthe hPSTI.SSImutant.M2 construction, with restriction sites shown. The signal sequence is underlined, the reactive site from SSI is shown in bold, and the M2 tag is in italics. Figure 10B depicts the amino acid sequence ofthe hPSTI.SSImutant modified subtilisin inhibitor without the M2 tag or the N- terminal alanine.

Figure 12 depicts the sequences inserted into pBN3 to make pBNppt, as described in the Examples. These are the signal sequence and prosequence of a subtilisin for use in cloning

Figure 13 depicts the sequence ofthe silkworm sequence, with a putative Pl- Pl' site identified.

Figure 14 depicts the rat agrin domain 5 sequence used to express in baculovirus. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel modified serine protease inhibitors, and in particular, novel modified subtilisin inhibitors. Generally, known human standard mechanism inhibitors do not exhibit suitable kinetic characteristics to allow their use in commercial processes involving subtilisin, although they do not generate undesirable immune responses. Similarly, known subtilisin inhibitors may have appropriate kinetic properties but are too allergenic or otherwise unsuitable for commercial use with humans. Accordingly, the present invention provides for the replacement ofthe specificity ofthe reactive site of the human standard mechanism inhibitor by residues which confer specificity to subtilisin. In this way, an unmodified human standard mechanism inhibitor is used as a "backbone", and the reactive site ofthe human standard mechanism inhibitor is recombinantly engineered to be more specific for subtilisin, thus forming an inhibitor specific for subtilisin without undesirable immunogenicity.

The human standard mechanism inhibitor may be modified in a variety of ways.

In one embodiment, all or part ofthe existing reactive site, which is specific for a protease other than subtilisin, is replaced with the reactive site of a known subtilisin inhibitor, such as an ovomucoid third domain, or Streptomyces subtilisin inhibitor (SSI), among others. Alternatively, the reactive site of a known subtilisin inhibitor is used as a prototype, and only selected residues are modified in the human standard mechanism inhibitor in order to confer proper specificity for subtilisin, as outlined below. In this embodiment, some ofthe original residues ofthe reactive site ofthe human standard mechanism inhibitor remain unchanged, and some are replaced by the residues present in the known subtilisin inhibitors. However, the new residues are sufficient to confer the appropriate subtilisin specificity to the human standard mechanism inhibitor Thus, the novel subtilisin inhibitors ofthe present invention comprise a modified human standard mechanism inhibitor, which has been recombinantly engineered to substitute at least one amino acid residue ofthe reactive site with a different amino acid to increase the specificity ofthe modified human standard mechanism inhibitor towards subtilisin.

By "subtilisin" herein is meant any number of serine endoproteases or protease variant ofthe "subtilase" family, preferably but not limited to alkaline serine endoprotease, produced by gram-positive bacteria or fungi, known in the art as subtilisin (Siezen et al., Protein Eng 4.719-737 (1991)) . A "target subtilisin" is a subtilisin which is inhibited by a modified subtilisin inhibitor ofthe present invention Examples of suitable subtilisins include, but are not limited to, the subtilisins from B. amyloliquefaciens, B. lichemformis, B. subtilis, and B. lentus, including subtilisin DY, subtilisin BPN', subtilisin Carsberg, subtilisin 168, subtilisin amylosaccharitius, mesentericopeptidase, subtilisin thermitase, and proteinase K

Also included within the definition of subtilisin are variant or mutant subtilisins A large number of variant subtilisins are known in the art For example, variants have been made which alter a number of characteristics ofthe enzyme, including pH stability and optima, thermostability, substrate specificity, and oxidative resistance See for example Estell, J Biotechnology 26 25-29 (1993), and Wells and Estell, Trends in Biochem Sci 13(8) 291-297 (1988) In general, a great deal is known about the substrate specificity of variants of subtilisin

The substrate specificity of any given subtilisin variant may be determined using a variety of synthetic substrates Specifically, subtilisin variants which may be inhibited with the novel inhibitors ofthe present invention include, but are not limited to, variants at the residue positions equivalent to the following positions in B. amyloliqυifaciens: Tyr6, Ser9, Ilel 1, Lysl2, Gnl9, Tyr 21, Thr22, Ser24, Asn25, Asp32, Ser33, Asp36, Ser37, Ser39, Ala45, Gly46, Ala48, Ser49, Met50, Ser53, Glu54, Thr55,

Asn56, Pro57, Phe58, His64, His67, Thr71, Asn76, Asn77, Ser87, Ser89, Lys94, Val95, Leu96, Gly97, Asp99, SerlOl, Glyl02, Glul03, Tyrl04, Ilel07, Glyl lO, Ilel 11, Ilel 15, Aspl20, Vall21, Ilel22, Metl24, Leul26, Glyl27, Glyl28, Prol29, Leul35, Glyl31, Leul35, Aspl40, Alal52, Alal53, Glyl54, Asnl55, Glul56, Glyl57, Thrl58, Serl59, GlylόO, Serlόl, Serl62, Serl63,

Thrl64, Vall65, Glyl66, Prol68, Glyl69, Lysl70, Tyrl71, Prol 72, Ilel75, Vall80, Serl82, Argl86, Alal87, Phel89, Serl91, Prol94, Glul95, Aspl97, Metl99, Ser204, Lys213, Tyr214, Gly215, Tyr217, Asn218, Ser221, Met222, His226, Ile234, Leu235, Ser236, Lys237, His238, Tφ241, Ser260, Phe261, Tyr262, Lys265, Ile268, or Gln275. See U.S. Reissue 34,606; WO 89/06279;

WO 92/10755; and 0 549 675 Bl, expressly incorporated herein by reference.

By "reactive site" herein is meant the region ofthe inhibitor with which the enzyme interacts during inhibition. That is, the reactive site ofthe inhibitor occupies the active site of its target enzyme. As is appreciated in the art, the exact location and size ofthe reactive site will vary with the inhibitor and protease to be inhibited. The reactive sites for a number of human standard mechanism inhibitors and subtilisin reactive sites for known subtilisin inhibitors are shown in the figures.

The reactive site generally comprises residues P10 to P10', where the Pl-Pl ' residues are the ones which occupy the S 1 -S 1 ' position ofthe enzyme's substrate (or inhibitor) binding site. That is, at least one amino acid within this region is altered, although, as outlined below, in preferred embodiments, more than one amino acid is substituted. Preferably, the residue or residues to be altered range between the P9 and P5' residues which occupy the S9 to S5' position ofthe enzyme binding site. In alternative embodiments, as outlined below, the residues from a smaller area ofthe reactive site are altered; for example, between P6 to P5', or P5 to P3 ', although it will be appreciated that any subsection of this area may be altered.

In a preferred embodiment, when the reactive site ofthe human standard mechanism inhibitor contains cysteine residues which form disulfide bridges, the cysteine residues are not altered, to maximize the inhibitor stability. It should be noted that in some cases, the subtilisin reactive site which is to replace the human standard mechanism inhibitor reactive site also contains cysteine residues at the identical positions. Thus, in the case where the entire reactive site ofthe human standard mechanism inhibitor is replaced by a subtilisin reactive site, the cysteine residues remain in the same location. In alternative embodiments, the cysteine residues may be removed.

By "subtilisin reactive site" herein is meant a reactive site, as defined above, in a known subtilisin inhibitor that is either specific for a target subtilisin or which inhibits a target subtilisin to a significant degree. That is, a subtilisin reactive site that inhibits only subtilisins is a subtilisin reactive site which is specific for subtilisin. Alternatively, a subtilisin reactive site in a known subtilisin inhibitor may significantly inhibit subtilisin, i.e. exhibit a low K, as is more fully discussed below, as well as inhibit other serine proteases

Known subtilisin inhibitors with the PIO to PIO' subtilisin reactive sites and Pl- Pl' sites shown are depicted in the Figures. Known subtilisin inhibitors with subtilisin reactive sites suitable for the use in the present invention include, but are not limited to, the third domains of avian ovomucoids and the Streptomyces subtilisin inhibitors (SSI). Included within the definition of SSI are the SSI-like inhibitors. Additionally, the sequence of a known subtilisin inhibitor from Bombyx mori (silkworm) has been determined, with a K, of approximately 10^"" to IO ¹³ (see Eguchi et al., J. Biochem. 115:881-884 (1994)), the sequence of which is shown in Figure 13. While the reactive site ofthe silkworm subtilisin inhibitor has not yet been reported, well known techniques may be used to determine the reactive site (see Ozawa et al., J. Biol. Chem. 241 :3955-3961

(1966) and Terabe et al., J. Biochem. 116: 1 156-1 163 (1994), both of which are hereby incoφorated by reference).

In addition to the ovomucoid third domains and the SSI subtilisin reactive sites, subtilisin reactive sites from other subtilisin inhibitors may be used. Generally speaking, a subtilisin with a suitable subtilisin reactive site has a K, for a subtilisin of IO^"6 or less, with 10"⁶ to IO ¹⁴ being suitable, and 10^"8 to 10 ^"l2 being preferred. Thus, for example, the reactive sites from the potato family of inhibitors, and the Bowman-Birk family of inhibitors may be used if the K,s are suitable (see Bode et al., Eur. J. Biochem. 204:433-451 (1992), incoφorated by reference).

In a preferred embodiment, the subtilisin reactive sites from avian ovomucoid third domains are used in the present invention. Avian ovomucoids generally comprise three domains, the last of which are well known as potent subtilisin inhibitors. The most commonly used ovomucoid for this puφose is the turkey ovomucoid third domain (abbreviated TOM or OMTKY.3), which has an approximate K, towards subtilisin of IO^"9 to 10^'10. The subtilisin reactive site of OMTKY.3 is a particularly preferred subtilisin reactive site in the invention.

The sequence of a large number of ovomucoid third domains are shown in Figure 5, with the P10 to P10' and Pl-Pl ' sites shown. In a preferred embodiment, the reactive site residues to be used comprise the P6 to P5' residues. Any ofthe subtilisin reactive sites ofthe ovomucoid third domain proteins depicted in Figure 5 may be used in the present invention. As outlined above, the ovomucoid third domains have cysteine residues in the P3 and P6' positions, which are generally conserved between the human standard mechanism inhibitors and the known subtilisin inhibitors.

In a preferred embodiment, the subtilisin reactive sites from Streptomyces subtilisin inhibitors (SSI) and "SSI-like" inhibitors, (collectively "SSI inhibitors") are used in the present invention. These include, but are not limited to, SSI from S. albogriseolus (see Obata et al., J. Biochem. 105:367-371 (1989)) which has an approximate K, for subtilisin in the range of IO^'12; the SSI- like (SIL) protein SILl of Kojima et al., Biochim. Biophys. Acta 1207: 120-125 (1994); the SIL2, SIL3 and SIL4 inhibitors of Taguchi et al., Eur. J. Biochem.

220:91 1-919 (1994); the Streptomyces lividans inhibitor (SLPI, not to be confused with the SLPI human standard mechanism inhibitor) of Ueda et al., J. Biochem. 112:204-21 1 (1992); see also STI-1 of Strickler et al., J. Biol. Chem. 267(5):3236-3241 (1992); STI-2 inhibitor of Strieker et al., supra; plasminostreptin (PSN) from S. antifibrinolyticus, Kakinuma et al., J. Biol.

Chem. 252(5): 1529- 1537 (1978); API-2c' inhibitor from S. griseoincamatus, Suzuki et al., Agric. Biol. Chem 45(3):629-634 (1981); the SIL8, SIL10, SIL13, and SIL14 inhibitors of Terabe et al., J. Biochem. 1 16: 1 156-1163 (1994), and the anticoagulant inhibitor STVCI, from Streptoverticillium cinnamoneum (J. Biochem. 1 15:752-761 (1994), all of which are hereby expressly incorporated by reference. Furthermore, Taguchi et al. Applied Environ. Microbio. 59(12):4338-4341 (1993), also expressly incorporated by reference, describe a number of SIL proteins from a variety of Streptomyces species. As for the ovomucoid third domains, the preferred reactive site residues ofthe SSI inhibitors for substitution into human standard mechanism inhibitors comprise the P6 to P5' residues. In addition, most, if not all, ofthe SSI inhibitors have cysteines in the P3 position, which allows the cysteine at this position to be kept constant. In addition, the reactive site of chelonianin is used as a subtilisin reactive site. The second domain of chelonianin, a two domain inhibitor ofthe red sea turtle, is highly homologous to MPI (see Seemuller et al., FEBS Lett. 199(l):43-48 (1986); and Kato et al., Fed. Proc. 38:832 (1979)). Chelonianin has been characterized as a subtilisin inhibitor. The PIO to PIO' subtilisin reactive site of chelonianin is shown in Figure 7 A, with the Pl-Pl' junction depicted. In a preferred embodiment, the chelonianin subtilisin reactive site replaces the reactive site of either domain ofthe MPI backbone.

In an additional embodiment, the reactive site of domain 5 of rat agrin is used as a subtilisin reactive site. The sequence of rat agrin domain 5 is shown in Figure

7B, with the PIO to PIO' subtilisin reactive site depicted. Rat agrin domain 5 has recently been shown to be a subtilisin inhibitor, with a K, in the IO^"5 to IO"⁶ range (data not shown).

Also included within the definition of "subtilisin reactive site" are modified, i.e. non-naturally occurring, reactive sites from known subtilisin inhibitors. That is, the subtilisin reactive sites of known subtilisin inhibitors may be recombinantly engineered as well to contain mutations within the subtilisin reactive site which are then exchanged with the reactive sites of human standard mechanism inhibitors to create the modified subtilisin inhibitors ofthe invention. For example, the Pl residue of turkey ovomucoid third domain has been changed from Leu 18 to Glu 18 to produce a potent inhibitor of Glu-specific Streptomyces griseus protease (Komiyama et al. J. Biol. Chem. 266(17): 10727- 10730 (1991)). In addition, mutants have been generated at the Pl position in SSI and found to retain specificity for subtilisin BPN' (Kojima et al., J. Bioche 109:377-382 (1991), expressly incoφorated by reference herein).

Thus, in a preferred embodiment, a mutant ofthe SSI reactive site may be used, as is depicted in Figure 11. This mutant reactive site is based on the SSI reactive site but contains Ala at P6, Ala at P5, Ala at P4, Leu at Pl, and Arg at P3^*.

As will be appreciated by those in the art, residues outside the reactive site may also interact with the enzyme, as is discussed below. These residues may also be substituted to optimize the interaction ofthe inhibitor and the subtilisin.

By "standard mechanism inhibitor" herein is meant an inhibitor which functions according to the standard mechanism outlined in Laskowski et al., Ann. Rev. Biochem. 49:593-626 (1980), hereby incoφorated by reference. This mechanism can be written as E + I *^*< L« C * X ■^*• L* *^*< E + I*

Where E is the enzyme, I* and I are modified inhibitor (reactive site peptide bond hydrolyzed) and virgin inhibitor (reactive site peptide bond intact) respectively, L and L* are loose, noncovalent (rapidly dissociable) complexes of E with I and I*, respectively, X is the relatively long-lived intermediate in the E+I*, and C is the stable enzyme-inhibitor complex Generally, the reactive site peptide bond is encompassed in at least one disulfide loop, which ensures that during conversion of virgin to modified inhibitor the two peptide chains cannot dissociate.

As is known in the art, standard mechanism inhibitors comprise several families of inhibitors, including the Kazal type inhibitors, the SSI type inhibitors, and the chelonianin family inhibitors. Included within the definition of human standard mechanism inhibitors, useful as backbones in the present invention, are the Kazal-type inhibitors such as PSTI, agrin and acrosin inhibitor (HUSI-II), and the chelonianin family inhibitors such as MPI. In a preferred embodiment, the definition of a standard mechanism inhibitor does not include the seφins or α₂- macroglobulin By "human standard mechanism inhibitor" herein is meant one ofthe standard mechanism inhibitors, as defined herein, which is from a human, or homologous human sequences which may not yet have been shown to proteinase inhibitors; that is, the naturally occuring amino acid sequence derived from humans.

Included within the definition of human standard mechanism inhibitor is human pancreatic secretory trypsin inhibitor (hPSTI) and PSTI-like inhibitors and sequences such as human agrin domain 5, human agrin domain 7, human acrosin inhibitor II, human hevin and human follistatin, and human mucous proteinase inhibitor (MPI) and MPI-like inhibitors, including skin-derived antileukoproteinase (SKALP; also known as human elafin precursor or ESI) and human epididymius protease inhibitor, HE4.

It should be understood that some ofthe unmodified human standard mechanism inhibitors may inhibit subtilisin without further modification. Thus, for example, unmodified hPSTI exhibits a K_t of roughly 10^'7 towards subtilisin. Accordingly, as will be more fully described below, the modified human standard mechanism inhibitors ofthe invention, i.e. the modified subtilisin inhibitors, have decreased K,s in relation to the unmodified human standard mechanism inhibitors.

It should be understood that some ofthe human standard mechanism inhibitors are comprised of a number of domains, not all of which may be relevant to the present invention. For example, as outlined below, agrin is a multidomain protein. The rat agrin sequence demonstrates nine tandem protease inhibitor domains, followed by a few unrelated domains. The sequence of human agrin protease inhibitor domain 7 is known, as depicted in Figure 3; however, other domains of agrin may also be used, if appropriate. Similarly, as outlined below, MPI contains two domains, either or both of which may be engineered as taught herein. HE4 also appears to be two domains.

It is important to note that the exact "start" ofthe protease inhibitor domain of a multidomain protein, including SKALP, MPI, agrin and the ovomucoid domains, may vary, as one skilled in the art will appreciate. That is, while the sequences depicted herein show specific N- and C-termini, one or more residues may be added or deleted without significantly affecting the present invention. In some cases, additional residues from the "linker" sequences between domains, or parts of other domains, may be included at the N- or C-terminus of a modified subtilisin inhibitor ofthe invention, as will be determined by those in the art. In fact, it may be possible to include an entire additional domain, or more, in the novel subtilisin inhibitors ofthe invention, although this is generally not preferred. That is, when the human standard mechanism inhibitor is a multidomain protein, two or more ofthe domains may be included, with at least one of these domains being recombinantly engineered to have an increased specificity for subtilisin, as outlined herein. In some cases, it may be possible to engineer two or more domains for subtilisin. Thus, for example, MPI has two domains, depicted as "1 " and "2" herein, both or either of which may be altered for increased subtilisin specificity. Similarly, HE4 appears to be two domains, either or both of which may be engineered as well.

In addition, the exact N- or C-termini ofthe inhibitors ofthe present invention may also vary somewhat, due to putative proteolytic processing Thus, for example, Szardenings et al., Protein Engineering 8(l):45-52 (1995), depicts the N-terminus of hPSTI as DSLGREA — . The Szardenings sequence was determined on the basis of protein sequencing. The N-terminus ofthe hPSTI of the examples has five additional N-terminal amino acids, GNTGADSLGREA — ; this sequence was obtained from the DNA sequence. It is possible to make inhibitors starting at any one of these positions, and all are included within the definition of hPSTI. This difference would appear to result from post¬ translational proteolytic processing.

Thus, the N- and C-termini ofthe novel subtilisin inhibitors ofthe invention may vary from those actually depicted herein, but the addition or deletion of a few residues, or even an entire domain, is not determinative.

In a preferred embodiment, the human standard mechanism inhibitor is hPSTI, and has the sequence depicted in Figure 3. The depicted reactive site to be altered comprises residues P6 to P5'. In particular, the residues which may be altered include Leu 13 (P6), Asn 14 (P5), Gly 15 (P4), Thr 17 (P2), Lys 18 (Pl), Ilel9 (Pl '), Tyr20 (P2'), Asp21 (P3'), and Val23 (P5') (amino acid numbering assumes DSLGREA as the N-teminus). In a preferred embodiment, Cysl 6 (P3) is not altered, since it forms a disulfide bridge within hPSTI which is putatively important for protein stability. Similarly, Cys24 (P6') is also not altered for the same reason, although in some embodiments these residues may be modified.

In addition, residues P5 to P9 may also be changed, since computer modelling suggests steric clashes with subtilisin at subtilisin residues 99-103. Further, Thr30 and Pro32 may also be replaced, since computer modelling suggests they closely approach subtilisin residues 155 and 156.

In a preferred embodiment, the human standard mechanism inhibitor is acrosin inhibitor II (GenBank accession number P20155). Acrosin inhibitor II has the sequence depicted in Figure 3, which portrays the putative Pl-Pl ' position and P10-P10' reactive site. The reactive site of acrosin inhibitor II was determined by alignment with the hPSTI sequence, and has been reported to inhibit acrosin and trypsin (see Moeritz et al., Gene 123:277-281 (1993)). In a preferred embodiment, the residues to be altered include the P6 to P5 ' residues, namely Leulό (P6), Prol7 (P5), Glylδ (P4), Pro20 (P2), Arg21 (Pl), Hιs22 (Pl '), Phe23 (P2'), Asn24 (P3'), Pro25 (P4') and Val26 (P5') (numbering based on GLFSK-- as the N-terminus) In a preferred embodiment, Cysl9 (P3) and Cys27 (P6') are not altered, due to putative disulfide bridge formation

In a preferred embodiment, the human standard mechanism inhibitor is a region of human agrin homologous to the PSTI-like domain 7 of rat agrin (see Rupp et al , J Neurosci 12 3535-3544 (1992), GenBank accession number S44195) The human domain 7 is depicted in Figure 3, and the Pl-Pl ' and putative reactive site (PIO to PIO') identified These sites were identified as for acrosin inhibitor II, by alignment with the hPSTI sequence In a preferred embodiment, the residues to be altered include, Val20 (Pl), Pro22 (P5), Arg23 (P4), Glu25 (P2), Hιs26 (Pl), Pro27 (Pl'), Pro28 (P2'), Hιs29 (P3'), Gly30 (P4'), Pro31 (P5"), Val32 (P6'), Gly33 (P8'), Ser34 (P9'), Asp35 (PIO'), and Gly36 (Pl 1')

(note numbeπng is from the sequence shown in Figure 3 shown for domain 7 only) In a preferred embodiment, the cysteines at positions P3, P6, P8 and P7' are not altered, although they may be if desired

In a preferred embodiment, the human standard mechanism inhibitor is human hevin protein, the sequence of which is depicted in Figure 3 with the putative reactive site depicted (see Girard et al , Immunity 2 113-123 (1995), Genbank accession number X82157 and X86693) In a preferred embodiment, the residues to be altered include, Val29 (PIO), Gln31 (P8), Asp32 (P7), Pro33 (P6), Val34 (P5), Thr35 (P4), Pro37 (P2), Pro38 (Pl), Thr35(Pl'), Lys40 (P2'), Pro41 (P3'), Leu42 (P4'), Asp43 (P5'), Gln44 (P6'), Val45 (PT), Gly47

(P9'), and Thr48 (PIO') (note numbering is from the sequence shown in Figure 3 only and is not necessarily from the N-terminus of hevin) In a preferred embodiment, the cysteines at positions P3, P9, and P8' are not altered, although they may be if desired.

In a preferred embodiment, the human standard mechanism inhibitor is human follistatin, the sequence of which is depicted in Figure3 with the putative reactive site depicted (Genbank accession numbers Ml 9480 and J03771 ). In a preferred embodiment, the residues to be altered include, Val29 (P8), Ala31 (P6), Pro32 (P5), Asp33 (P4), Ser35 (P2), Asn36 (Pl), Ile37 (Pl'), Thr38 (P2'), Tφ39 (P3'), Lys40 (P4'), Gly41 (P5'), Pro42 (P6')_; Val43 (PT), Gly45 (P9'), and Leu46 (PIO') (note numbering is from the sequence shown in Figure 3 only and is not necessarily from the N-terminus of follistatin). In a preferred embodiment, the cysteines at positions P3, P7, P9, and P8' are not altered, although they may be if desired.

In a preferred embodiment, the human standard mechanism inhibitor is MPI. Mucous protease inhibitors have been identified in humans and called a variety of names, including secretory leukocyte protease inhibitor (SLPI), bronchial mucous inhibitor (BMI), cervical uteri secretion inhibitor (CUSI), bronchial leukocyte protease inhibitor (BLPI), and human seminal secretion inhibitor (HUSI-I, also called antileukoprotease). To date, it appears that all of these are derived from a single polypeptide with possible different proteolytic processing at the N-terminus (see Grutter et al., EMBO J. 7(2):345-351 (1988); but see

Klasen et al., Biochem. Biophys. Res. Comm. 128(l):285-289 (1985))

The sequence of MPI (identical to HUSI-I) is depicted in Figure 4, with the Pl- Pl ' sites and the P10-P10' reactive sites shown. As outlined above, MPI appears to have two domains, arbitrarily designated in Figure 4 as the " 1 " domain and the "2" domain. Either or both ofthe domains find use in the present invention. That is, in one embodiment the reactive site of only a single domain is altered, while the other domain is left unchanged. Alternatively, one domain is altered and the other is deleted. In yet another embodiment, both domains are altered.

In the "1" domain, the residues that will be altered include Ser 15, Alal5, Glnl7, Leu 19, Arg20, Tyr21, Lys22, Lys23, Pro24, (reactive site inferred from domain 2) and Glu25, with Cys 18 and Cys26 remaining unchanged in a preferred embodiment.

In the "2" domain, the residues to be altered include Tyr68 (P5), Gly69 (P4), Gln70 (P3), Leu72 (Pl), Met73 (Pl'), Leu74 (P2'), Asn75 (P3'), Pro76 (P4'), Pro77 (P5'), Gln78 (P6') and Phe79 (P7'), with Cys71 (P2) and Cys80 (P8') remaining unchanged in a preferred embodiment.

In a preferred embodiment, the human standard mechanism inhibitor is a domain of human skin-derived antileukoproteinase (SKALP). SKALP has also been called human elafin precursor (ELAFIN) and residues 68 to 1 17 of SKALP comprise the protease inhibitor domain that exhibits a 40% homology to MPI The sequence of one domain of SKALP is shown in Figure 4, with the putative reactive site PIO to PIO' and the Pl-Pl ' junction displayed

In a preferred embodiment, the residues to be altered include the P6 to P5' residues, namely Ilel4 (P6), Leul5 (P5), Ilelό (P4), Argl 7 (P3), Alal9 (Pl), Met20 (Pl'), Leu21 (P2'), Asn22 (P3'), Pro23 (P4'), Pro24 (P5'), Asn25 (P6') and Arg26 (P7' (note numbering is from sequence depicted in Figure 4 and is not from the N-terminus ofthe complete SKALP protein). In a preferred embodiment, Cysl 7 (P2) and Cys26 (P8') are not altered, due to putative disulfide bridge formation.

In an additional embodiment, the human standard mechanism inhibitor is a human epididymis protease inhibitor (HE4; see Kirchhoff et al., Biol. Reprod. 45:350-357 (1991). The sequence of HE4 is depicted in Figure 4, with the putative P10-P10' reactive site and the Pl-Pl ' site shown. It should be understood that the precise reactive site of either domain of HE4 (nor MPI-1 and SKALP) is not currently known, although it may be determined by those in the art as outlined above for silkworm, and thus the site depicted in Figure 4 is one of several possibilities. HE4 is also putatively comprises two domains, with the "1" and "2" domains depicted in Figure 4.

As for the other human standard mechanism inhibitors, the reactive site of HE4 may be altered. In the "1" domain, the residues to be altered include the P6 to P6' residues, namely Alal5 (P6), Asplό (P5), Glnl7 (P4), Asnlδ (P3), Thr20

(Pl), Gln21 (Pl^*), Glu22 (P2'), Val24 (P4'), Ser25 (P5') and Asp26 (P60- In a preferred embodiment, Cysl 9 (P2) and Cys23 (P3 are not altered, due to putative disulfide bridge formation. It should be noted that the actual amino acids at the P6-P6' positions may be different if the Pl-Pl' junction is shifted.

In the "2" domain, the residues to be altered include the P6 to P6' residues, namely Gln64 (P6), Leu65 (P5), Gly66 (P4), Leu67 (P3), Arg69 (Pl), Asp70 (PI O, Gln71 (P20, Gln73 (P40, Val74 (P5') and Asp75 (P6'). In a preferred embodiment, Cys68 (P2) and Cys72 (P3 ') are not altered, due to putative disulfide bridge formation.

Once the unmodified human standard mechanism inhibitor to be used as the backbone for the modified subtilisin inhibitors ofthe present invention is selected, a reactive site which is particularly effective against subtilisin ("subtilisin reactive site") is constructed to replace the human standard mechanism inhibitor reactive site. This engineering ofthe human standard mechanism inhibitor to contain a subtilisin reactive site may be done in several ways. In one embodiment, part or all ofthe human standard mechanism inhibitor reactive site may be replaced. That is, the PIO to PIO' residues may be excised and replaced with the PIO to PIO' residues of a subtilisin reactive site from a known subtilisin inhibitor. Alternatively, in a preferred embodiment, direct replacement of less than the entire reactive site may be accomplished. For example, the P6 to P5' residues may be exchanged. In a preferred embodiment, there is no "replacement" per se; rather, only selected residues within the reactive site ofthe human standard mechanism inhibitor are substituted by the appropriate residues at the equivalent position in the subtilisin reactive site, thus using the subtilisin reactive site from a known subtilisin inhibitor as a model or prototype.

In a preferred embodiment, the reactive site residues of the unmodified human standard mechanism inhibitor are replaced with the reactive site residues of a known subtilisin inhibitor, to generate a modified subtilisin inhibitor. As discussed above, this may be done using the entire reactive site (PIO to PIO'), or portions ofthe reactive site (preferably P6 to P5

For example, in a preferred embodiment, the P6 to P5' residues of hPSTI are replaced with the P6 to P5' residues of an ovomucoid third domain protein, preferably OMTKY.3, or the P6 to P5' residues of an SSI protein, preferably the SSI from S. albogriseolus.

Preferred standard mechanism inhibitors/subtilisin reactive site pairs include hPSTI/OMTKY3, hPSTI/ovomucoid, hPSTI/SSI, MPI-1 or 2/red sea turtle inhibitor, MPI/OMTKY3, and MPI/SSI, although those in the art will appreciate that any combination of backbone and subtilisin reactive site may be used

In an altemate embodiment, only selected residues within the human standard mechanism inhibitor reactive site are altered. This is done using a subtilisin reactive site as a model or prototype. Thus, for example, at least one amino acid ofthe human standard mechanism inhibitor reactive site is substituted with the amino acid at the equivalent position in the subtilisin reactive site For example, the lysine at the Pl position of hPSTI may be substituted with a leucine, which is the Pl residue of OMTKY.3, or a methionine, which is the Pl position of SSI. In other embodiments, more than two, three, four or five residues may be altered, with at least three being preferred and at least five being particularly preferred.

In order to select which residues should be substituted, one of two methods may be employed. If the structure ofthe inhibitor and/or reactive site is known, modeling may be done as is known in the art Alternatively, each amino acid residue ofthe reactive site may be changed to each ofthe other amino acids, or a subset ofthe amino acids, and the resulting mutants screened, as is known in the art Routine experimental techniques are available to easily mutate and screen the backbones In general, the Pl, P2 and P4 residues ofthe human standard mechanism inhibitor are altered, since subtilisin has distinct binding pockets at the Sl, S2 and S4 sites corresponding to these sites Thus, these three residues within the human standard mechanism may be altered first

In addition, the characteristics ofthe target subtilisin may be useful in constructing a modified subtilisin inhibitor That is, inhibitors can be designed to mimic the substrate specificity ofthe target subtilisin, a process that is known in the art See for example, Lu et al , J. Biol Chem 268(20) 14583-14585 (1993), Komiyama et al , J. Biol. Chem. 266(17): 10727 (1991)

The modified subtilisin inhibitors ofthe present invention are constructed such that the kinetic parameters ofthe modified inhibitor are altered relative to the unmodified inhibitor This may be quantified in several ways In a preferred embodiment, the dissociation constant for inhibition of a subtilisin by the modified subtilisin inhibitor is lower than the dissociation constant ofthe unmodified human standard mechanism inhibitor. As noted above, some unmodified human standard mechanism inhibitors exhibit some level of inhibition of subtilisin, although it is generally not significant enough for practical applications. Accordingly, what is important in the present invention is that the dissociation constant for subtilisin decreases as a result ofthe engineering ofthe reactive site to be more specific for subtilisin.

Accordingly, in a preferred embodiment, the modified human standard mechanism inhibitors ofthe invention have dissociation constants for at least one subtilisin that is decreased by at least a factor of 10 relative to the unmodified human standard mechanism inhibitor. In a preferred embodiment, the dissociation constant is decreased by a factor of 100, or IO^"2, with IO^"3, 10^"4, 10^"5 and 10^"6 also possible. A decrease of at least IO^"2 is preferred.

By the term "inhibition constant" or "dissociation constant" or grammatical equivalents herein is meant the equilibrium dissociation constant for the reversible association of inhibitor with enzyme. The dissociation constant for inhibition of a protease with the inhibitor is generally referred to by those in the art as K,. The dissociation constants are defined and determined as below

The determination of dissociation constants is known in the art. For example, for reversible inhibition reactions such as those ofthe present invention, the reaction scheme is as follows:

Equation 1

E + I * E- 1 The enzyme and the inhibitor combine to give an enzyme-inhibitor complex, E-I. This step is assumed to be rapid and reversible, with no chemical changes taking place; the enzyme and the inhibitor are held together by non-covalent forces. In this reaction, kj is the second order rate constant for the formation ofthe E-I reversible complex, i.e. the "on" rate, referred to herein as "k,.-". k₂ is the first order rate constant for the disassociation ofthe reversible E-I complex, i.e. the "off' rate, referred to herein as "k_off". In this reaction, K, = k_j/k,.

The measurement ofthe equilibrium constant K[ proceeds according to techniques well known in the art, as described in the examples. For example, assays generally use synthetic chromogenic or fluorogenic substrates.

Particularly preferred are methods for the determination of K,s for tight -binding inhibitors. The K, may be determined in several ways, as outlined below.

In one embodiment, the K, is determined as a ratio of rate constants, as outlined in Tonomura et al., in Protein Protease Inhibitor - The Case of Streptomyces Subtilisin Inhibitor, Hiromi et al., ed., Elseview Science Publishers B.V., 1985, chapter 9, particularly pages 297-299. Briefly, the K, is determined as the ratio ofthe k,-. and k_ofr rates, which may be determined either directly or graphically. This method is outlined in Example 2

The K, for competitive binding inhibitors may also be determined from the following calculation:

Equation 2

[i { l-(v v_o)} = {K_I/(v/v₀)} + [E]_o wherein

[I]₀ is total inhibitor concentration, [E]₀ is the total enzyme concentration, v₀ is the rate of substrate hydrolysis in the absence of inhibitor, and Vj is the rate in the presence of competitive inhibitor. A plot of [I]_</{ l-(v/v } vs. v v_t gives a slope equal to K,(l + [SJ/K-.), which is the apparent K_τ, with an x-intercept of [E]₀.

It is to be understood that dissociation constants are a particularly useful way of quantifying the efficiency of an enzyme with a particular substrate or inhibitor, and are frequently used in the art as such. If an inhibitor exhibits a very low K,, it is an efficient inhibitor. Accordingly, in a preferred embodiment, the K, ranges from IO^"2 to IO^"12, with IO^"4 to IO^'10 being preferred, and 10^'7 to 10^"10 being particularly preferred. However, the K, should not be so low as to prevent the release ofthe inhibitor from the subtilisin enzyme upon dilution, as is outlined below. Thus, generally, K,s of less than IO^"10 are not preferred, although K,s in this range are acceptable if the k_off is fast enough.

In addition, in a preferred embodiment, the modified subtilisin inhibitors ofthe invention have dissociation constants which are low enough to allow adequate and efficient inhibition ofthe subtilisin in a concentrated form or in a detergent formulation, yet have k_off rates which allow fast release ofthe inhibitor upon dilution ofthe detergent or formulation.

Accordingly, in a preferred embodiment, the modified subtilisin inhibitors preferably have k_off rates which reflect fast half times of dissociation (half time of dissociation = 0.693/k_o(T). Preferred half times of association range from about 15 seconds to about 300 seconds, or k_offs ranging from about 0.0462 to about 0.00231. However, as will be appreciated by those in the art, there is a balance between fast half times of dissociation and the K, The k, and k₂, ie. k_m and k_off constants are determined using well known techniques, as is generally described above and in the Examples In one embodiment, when the inhibitor is to be used as part of an enzyme and detergent composition, the ratio ofthe dissociation constant to the protease concentration is in the range from 0.006 to 6.

The Kj may be also be determined by measuring the 1^ (or IC₅₀₎. Generally, IC_JQ is the concentration of inhibitor at which the enzyme reaction velocity is 50% of the uninhibited reaction. When the enzyme is preincubated with a tight-binding inhibitor, the K, may be determined via Equation 3 :

IC₅₀ = 1/2E, + K,

where E, is total enzyme concentration in molar. IC^s are determined according to Cha et al., Biochem. Pharmacology 24:2187-2197 (1975); see also

Cha et al. Biochem. Pharmacology 24:2177-2185 (1975), both of which are incoφorated by reference.

As for K,, IC^ measurements are a useful way of quantifying the efficiency of an inhibitor. The lower the IC_so, the more efficient the inhibitor

The assays for the determination of kinetic parameters are done as is known in the art, as is shown in the Examples For example, the reactions are usually conducted at the pH optimum ofthe subtilisin enzyme, in the presence of small concentrations of Triton X-100 or other detergents, as subtilisin tends to stick to disposable cuvettes and microcentrifuge.

In a preferred embodiment, the human standard mechanism inhibitor is hPSTI, and the subtilisin reactive site is from an ovomucoid third domain. A particularly preferred subtilisin reactive site is the turkey ovomucoid third domain subtilisin reactive site. The sequence ofthe modified subtilisin inhibitor of this embodiment is shown in Figure 10 In a preferred embodiment, the human standard mechanism inhibitor is hPSTI, and the subtilisin reactive site is from a SSI inhibitor. A particularly preferred subtilisin reactive site is the SSI subtilisin reactive site from S. albogriseolus. The sequence ofthe modified subtilisin inhibitor of this embodiment is shown in Figure 9.

In a preferred embodiment, the human standard mechanism inhibitor is MPI, and the subtilisin reactive site is from either domain of chelonianin from red sea turtle. Similarly, MPI with the reactive site from OMTKY.3 or a SSI is also preferred.

Once the subtilisin reactive site and the human standard mechanism inhibitor are chosen, the nucleic acids encoding the modified subtilisin inhibitors ofthe present invention are made using techniques well known in the art. As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA, mRNA and oligonucleotides including sense and anti-sense nucleic acids. Generally, the nucleic acid encoding the human standard mechanism inhibitor is used as the starting material.

However, since the inhibitors ofthe invention are not large, it is also possible to construct the nucleic acids encoding the inhibitors de novo, using synthetic techniques well known in the art.

These techniques are generally outlined in the examples, and may be used when the entire reactive site, a portion of the reactive site or only selected residues within the reactive site ofthe human standard mechanism inhibitor are to be replaced. /1

-29-

As it is understood by those in the art, once the amino acid sequence of a modified subtilisin inhibitor is selected, it is possible to generate a large number of nucleic acids which code for the modified subtilisin inhibitor amino acid sequence, due to the degeneracy ofthe genetic code. Thus, for example, synthetic oligonucleotides may be made which use different codons than the naturally occurring or native codons, and the synthetic oligonucleotides incoφorated into the modified subtilisin inhibitor nucleic acid using techniques well known in the art. In a preferred embodiment, the nucleic acid mimics as closely as possible the human naturally occuring sequence ofthe human standard mechanism inhibitor.

In addition, the nucleic acids encoding the modified subtilisin inhibitors ofthe present invention may also include additional coding regions. For example, the nucleic acids may include nucleotides which encode sequences useful in purification or identification, such as epitope or affinity tags such as M2, as outlined in the Examples. The M2 tags ofthe examples were all attached at the

C-terminus, but N-terminal tags may be used as well (data not shown). Similarly, as outlined above, additional N- or C-terminal amino acid residues may be encoded, or additional domains.

Once the nucleic acids encoding the modified subtilisin inhibitors ofthe invention are made, they are cloned and expressed in a variety of host cells for additional manipulations and expression.

The expression vector and host cell will depend on the end use ofthe modified subtilisin inhibitor. In a preferred embodiment, the modified subtilisin inhibitors are produced in host cells, purified or concentrated as needed, and then added to subtilisin-containing compositions such as detergent formulations, as outlined below. In this embodiment, the modified subtilisin inhibitor may be produced in a variety of host cell types, as outlined below. Alternatively, the modified subtilisin inhibitor may be co-expressed with the target subtilisin in the host cell, to eliminate the need for two fermentations. An additional advantage is that the yield of target subtilisin may also increase due to lowered autolysis.

Using the nucleic acids ofthe present invention which encode the modified subtilisin inhibitors ofthe invention, a variety of expression vectors are made.

The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Particularly preferred are integration vectors for integration into subtilisin production strains, as outlined below.

Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the modified subtilisin inhibitor. "Operably linked" in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence ofthe modified subtilisin inhibitor in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5 ' to the modified subtilisin inhibitor coding region The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the modified subtilisin inhibitor; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the modified subtilisin inhibitor in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.

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

Promoter sequences encode either constitutive or inducible promoters The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.

In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.

Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating and replicating vectors are well known in the art.

In addition, 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

The modified subtilisin inhibitors ofthe present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a modified subtilisin inhibitor, under the appropriate conditions to induce or cause expression ofthe modified subtilisin inhibitor. The conditions appropriate for modified subtilisin inhibitor expression will vary with the choice ofthe expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation ofthe host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing ofthe harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus species such as B. subtilis and B. licheniformis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, Streptomyces species such as S. lividans, fungal strains such as Aspergillus, Tricoderma and others, immortalized mammalian myeloid, and lymphoid cell lines.

When the modified subtilisin inhibitor is produced independently from the target subtilisin, particularly preferred expression systems include Bacillus species such as B. subtilis.

When the modified subtilisin inhibitor is to be produced simultaneously with the target subtilisin, particularly preferred expression systems include Bacillus species such as B. subtilis.

In a preferred embodiment, modified subtilisin inhibitors are expressed in bacterial systems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of modified subtilisin inhibitor into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end ofthe coding sequence This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include 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 may also be used and are known in the art In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid ofthe trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable In E. coli, the ribosome binding site is called the Shine-

Dalgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream ofthe initiation codon

The expression vector may also include a signal peptide sequence that provides for secretion ofthe modified subtilisin inhibitor in bacteria The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion ofthe protein from the cell, as is well known in the art The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane ofthe cell (gram-negative bacteria) The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed Suitable selection genes include genes which render the bacteria resistant 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 assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.

The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.

In one embodiment, modified subtilisin inhibitors are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.

Mammalian expression systems are also known in the art and are used in one embodiment. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for modified subtilisin inhibitor into mRNA. A promoter will have a transcription initiating region, which is usually place proximal to the 5' end ofthe coding sequence, and a TATA box, located 25-30 base pairs upstream ofthe transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site A mammalian promoter will also contain an upstream promoter element, typically located within 100 to 200 base pairs upstream ofthe TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, and heφes simplex virus promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3 ' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus ofthe mature mRNA is formed by site-specific post¬ translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection ofthe DNA into nuclei.

In a preferred embodiment, stably transfected mammalian cell lines which express modified subtilisin inhibitors are made

In a preferred embodiment, modified subtilisin inhibitor is produced in yeast cells Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C_ maltosa. Hansenula polymoφha. Kluyveromyces fragilis and K lactis. Pichia guillerimondii and P pastoris. Schizosaccharomyces po be. and Yarrowia lipolytica Preferred promoter sequences for expression in yeast include the inducible GAL 1,10 promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3- phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3- phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene. Yeast selectable markers include ADE2, HIS4, LEU2, TRPl, and ALG7, which confers resistance to tunicamycin; the G418 resistance gene, which confers resistance to G418; and the CUPl gene, which allows yeast to grow in the presence of copper ions

A recombinant modified subtilisin inhibitor may be expressed intracellularly or secreted.

The modified subtilisin inhibitor may also be made as a fusion protein, using techniques well known in the art. The modified subtilisin inhibitor may be made as a fusion protein to increase expression. For example, the modified subtilisin inhibitor is made as a fusion protein with the subtilisin to be inhibited, preferably using a linker cleavable by the subtilisin or another enzyme present in the mixture Once the fusion protein is secreted, the active subtilisin will cleave the linker, releasing the inhibitor, which then inhibits the subtilisin from further proteolytic activity

In this embodiment, any number of suitable linkers may be used Peptide linkers are preferred, with linkers of about 3 to about 10 amino acids being preferred, although longer linkers may be used Particularly preferred are linkers of homoalanine, such as peptides of five alanine residues

The modified subtilisin inhibitors ofthe invention may also be translationally coupled to another protein, as is known in the art

As discussed above, amino acid residues ofthe human standard mechanism inhibitor other than those ofthe reactive site may also be altered In this way, substitutional, insertional or deletional variants outside ofthe reactive site of human standard mechanism inhibitors are made These modifications are ordinarily prepared in the same way as the reactive site substitutions Thus, site specific mutagenesis of nucleotides in the DNA encoding the modified subtilisin inhibitor is done, using cassette mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. Alternatively, the proteins or the nucleic acids may be prepared by in vitro amino acid or nucleic acid synthesis using established techniques. Alterations in positions outside ofthe reactive site typically do not alter the qualitative biological activity ofthe unmodified human standard mechanism inhibitor, and preferably do not increase the allergenicity ofthe inhibitor. In a preferred embodiment, the human standard mechanism inhibitor is altered such that any subtilisin proteolytic sites are removed. That is, the target subtilisin may cleave the human standard mechanism inhibitor outside ofthe reactive site, resulting in a loss of activity of the inhibitor. These secondary proteolytic sites may be altered to decrease or eliminate this proteolytic cleavage.

In a preferred embodiment, the modified subtilisin inhibitor is purified or isolated after expression. Modified subtilisin inhibitors may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample, standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase

HPLC chromatography, and chromatofocusing. For example, the modified subtilisin inhibitor may be purified using a standard antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful, particularly when the modified subtilisin inhibitor is produced with the target subtilisin. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use ofthe modified subtilisin inhibitor. In some instances no purification will be necessary. In a preferred embodiment, the modified subtilisin inhibitors ofthe invention are co-expressed with the target subtilisin, and are subjected to whatever downstream processing the subtilisin normally receives In this embodiment, it is expected that the inhibitor will form an enzyme/inhibitor complex with the subtilisin, and thus the two will be relatively inseparable while in a concentrated form. Thus for example, fermentation broth may be concentrated and subjected to a diafiltration and ultrafiltration step, a crystallization step, granularization or standard chromatographic procedures

Once expressed and purified if necessary, the modified subtilisin inhibitors are useful in a number of applications

In one embodiment, the modified subtilisin inhibitors are used as protease inhibitors in a variety of uses For example, the inhibitors are coupled to solid supports for use as chromatography columns for the purification of subtilisin The inhibitors may be used to titrate the active sites of subtilisin for use in quantifying the amount of subtilisin present, for example as a diagnostic tool

In a preferred embodiment, the modified subtilisin inhibitors are incorporated into detergent formulations containing subtilisin enzymes Detergent formulations are well known in the art The inhibitors are combined with detergents, builders, bleach and/or fluorescent whitening agents as is known in the art for use with proteolytic enzymes The inhibitors find particular use in formulations containing bleaching or oxidizing agents such as perborate, percarbonate or hydrogen peroxide, as the inhibitor can help stabilize the enzyme Suitable detergents include linear alkyl benzene sulfonates, alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linear alcohol

The inhibitors may be incoφorated into liquid, powder or granular form See for example U S Re 34,606, EP 0 549 675 Bl, U S Pat Nos 5,039,446, 5,178,789, all of which are expressly incorporated herein, and references cited therein.

When used in a detergent formulation, the modified subtilisin inhibitor is added in an amount at least about 70 percent on a molar basis ofthe enzyme to be stabilized, with at least about 80% to about 100% being preferred. The modified subtilisin inhibitor is added to the concentrated detergent or cleaning composition in an amount proportional to the inhibition constant (K,). Thus, in a preferred embodiment, at least about 55% ofthe target subtilisin ofthe detergent is essentially bound to the inhibitor in the concentrated form, with at least about 60% being preferred and at least about 70% being particularly preferred. However, when the detergent is diluted with water or other appropriate liquid, less than 45% ofthe enzyme is inhibited with the modified subtilisin inhibitors ofthe invention. Generally this dilution is from 2 to about 10,000 or 100,000 times, with a 670 fold dilution being standard in laundry applications. In a preferred embodiment, less than about 30 % ofthe enzyme is inhibited upon dilution, with less than about 20% being particularly preferred. The percentage ofthe enzyme-inhibitor complex may be determined using Equation 1.

In a preferred embodiment, human subjects exhibit little or no immune response upon exposure to the modified subtilisin inhibitors ofthe present invention.

That is, the modified subtilisin inhibitors have significantly reduced allergenicity as compared to known subtilisin inhibitors.

The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects ofthe invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative puφoses. All references cited herein are incoφorated by reference.

EXAMPLES Example 1 Cloning and Expression of Modified Subtilisin Inhibitors

Inhibitors may be produced in the appropriate organism using any well established method (for example, see Methods Enzymol. 185. 1990, the entire volume). In general, the expression system will require an appropriate transcriptional promoter, preferably inducible, upstream ofthe inhibitor gene with standard transcriptional termination sequences placed downstream ofthe gene. A ribosome binding site (Stormo, G.D., Schneider, T.K., and Gold, L.M., Nucleic Acids Res. K), 2971-2996, 1982) or translational initiation sequences (Kozak, M., Cell 44, 283-292, 1986) will be needed for expression in prokaryotes and eukaryotes, respectively. The inhibitor gene may contain extra coding sequences at either end that simplify the gene construction or aid in the purification ofthe final protein product. For extracellular production, a signal sequence (von Heijne, G., Genetic Engineering 14, 1-1 1 , 1992) may be fused to the 5' end ofthe inhibitor gene. The inhibitor gene may also be fused at either end to a much larger protein gene (e.g. to produce an inhibitor protein fused to the C-terminal end of subtilisin) The entire expression system would be introduced into an integrational or independently replicating vector suitable to the chosen organism. Readily available transformation or transfection methods could be used to introduce the expression vector into a given host Well characterized selection methods could be used to isolate and maintain cultures that carry the desired vector. Standard protocols would be used for the growth ofthe organism and induction ofthe expression ofthe inhibitor gene The inhibitor gene may be coexpressed with another protein gene (e.g. subtilisin) if desired For use in industrial products, the preferred organism for the expression ofthe inhibitors would be a Bacillus species, typically Bacillus subtilis. The use of various host strains, vectors, promoters, terminators, translational signals, signal sequences, and growth conditions for the production of heterologous proteins in Bacillus subtilis has been well documented in the literature (see Wang, L -F., and Doi, R.H., Biotechnology 22, 63-104, 1992 and references within). Furthermore, the molecular genetics and physiology of Bacillus subtilis has been studied in detail (see relevant chapters in Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics, L. Sonenshein, J.A. Hoch, and R. Losick, eds., American Society for

Microbiology, D.C, 1993).

Specific examples

Inhibitor gene construction

The inhibitor genes were synthesized by using PCR (polymerase chain reaction) to amplify overlapping synthetic oligonucleotides (synthesized using an ABI 394

DNA/RNA Synthesizer). Appropriate restriction sites and M2 tag purification sequences were added to the inhibitor gene sequences to simplify cloning and protein purification, respectively (see Figures). The DNA sequence was verified using a 373A DNA Sequencer and a Cycle Sequencing kit (ABI).

E. coli expression

The phoA promoter and signal sequence was amplified from E. coli MM294 cells by using PCR. The PCR fragment was digested with Bglll and BssHII and purified from a 5% polyacrylamide gel. An inhibitor gene, as a BssHU-Pstl fragment, was ligated with the promoter fragment into the vector pKK223-3 (Pharmacia) that had been previously cleaved with BamHI and Psl (Figure 12).

Different reactive sites were introduced by utilizing the .4ιτII and Kpnl restriction sites (Figures). The inhibitor expression vectors (e.g. pKKρhoA.PSTI.SSI.M2, Figure 2) were used to transform E. coli NL106 cells Cultures were grown overnight in low phosphate media (Yuan, L , Craig, S.P., McKerrow, J.H., and Wang, C C, al, J Biol Chem 265, 13528-13532, 1990) for the induction of the phoA promoter Significant levels ofthe inhibitor were produced in the extracellular medium

B. subtilis expression

The replicating plasmid, pBN3 (Figure 1), is an E. coh-B. subtilis shuttle vector constructed from pBR322 (Bolivar, F , Rodriguez, R L , Greene, P J., Betlach, M.C , Heyneker, H.L , and Boyer, H W., Gene 2, 95-1 13, 1977) and pUBl 10 (Gryczan, T J , Contente, S , and Dubnau, D , J Bacteriol 134, 318-329, 1978) with a polycloning site added using a synthetic oligonucleotide The original EcoRI site in pBR322 was eliminated by filling in the site with Klenow enzyme and ligating the resultant blunt ends The PstI site in the ampicillin resistance gene of pBR322 was removed by swapping the Fspl-Aatll fragment from the original plasmid with one from pUCBM21 (Boeringer Mannheim) The pBNppt expression vector was constructed by inserting four PCR amplified fragments into the EcoRI and Hindlll sites of pBN3 (Figure 1) The B. subtilis aprE promoter was amplified from B. subtilis BG2036 cells as an Eco J-BamHl fragment Sequences from the B. amylo quefaciens subtilisin gene were amplified from pS4 5 (Well, J A , Ferrari, Ε , Henner, D J , Estell,

D A , and Chen, E Y , Nucleic Acids Res H, 791 1-7925, 1983) The signal sequence region was isolated as a BcH-BssHll fragment, the pro sequence region as a BssHll-Sphl fragment, and the terminator region as a Sphl-Hindlll fragment (Figure 1) Inhibitor genes were then inserted into the pBNppt expression vector utilizing the -955HII and BamHI sites The starvation method

(Anagnostopoulos, C , and Spizizen, J , J Bacteriol 8J_, 741 -746, 1961 ) was used to transformation B. subtilis WB600 with the inhibitor expression vectors and resistance to neomycin was used for selection The inhibitors were produced in flasks by shaking (225 rpm) overnight at 37°C in very rich medium (Halling, S.M., Sanchez-Anzaldo, F.J., Fukuda, R., Doi, R.H., and Meares, C.F., Biochemistry 16, 2880-2884, 1977).

Baculovirus expression

The PCR amplified genes for PSTI.M2 and for domain 5 of rat agrin were cloned into the baculovirus transfer vector pAcGP67 A (PharMingen) as

BamHl-EcόRl fragments (Figures 8 and 14). Spodoptera frugiperda (Sf9) were transfected and inhibitor proteins produced as specified by the manufacturer (PharMingen).

Example 2 Kinetic Characterization

Unless otherwise noted, all materials used were of reagent grade or better.

Isolation of PSTLSSI (PSTI scaffold with Streptomyces subtilisin inhibitor reactive site).

One liter of fermentation broth of pKKphoA.PSTI.SSI..m2 was centrifuged at 10,000 φm in a Sorvall RC-5A centrifuge for ten minutes to pellet the cells.

The supernatant was removed and filtered using a 0.45μm filter. Immediately after filtering PMSF (phenylmethylsulfonylfluoride, Sigma Chemical Co., St. Louis, MO) and EDTA (ethylenedinitrolotetraacetic acid, Sigma Chemical Co., St. Louis, MO) were each added to a final concentration of ImM. The resultant supernatant was then adjusted to pH 7.0 with a ION NaOH solution. Since

PSTLSSI possesses a M2-flag peptide on the C-terminus, 10 ml of anti-FLAG M2 affinity resin (Eastman Kodak Co., New Haven, CT) were added to the supernatant. This suspension was gently stirred for 1 h at room temperature after which the resin with PSTI. SSI. M2 attached was filtered using a 0.8μm filter (Baxter, Hayward, CA). The resin was resuspended in 5mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, J.T. Baker, Phillipsburg, NJ), 150mM NaCl, pH 7.0 and poured into a XK-16 FPLC column (Pharmacia, Upsala, Sweden). The protein was eluted from the column in the following manner: The column was first washed with 5mM HEPES, 150mM NaCl, pH 7.0 buffer until the optical density at 280 nm ofthe effluent reached baseline level. Next the column was washed with 5mM HEPES, IM NaCl, pH 7.0 buffer to remove any non-specifically bound proteins. Finally the PSTI.SSI.m2 was eluted in 4 column volumes of lOOmM sodium acetate, IM NaCl, pH 4.5 buffer. Fractions containing inhibitiory activity against subtilisin A (CalBiochem, La Jolla, CA), were pooled, adjusted to pH 7.0 with IM TRIS buffer, pH 8.0 (tris(hydroxymethyl)aminomethane. No further purification or concentration ofthe protein was necessary for analyses

Determination of Concentration of PSTI.OMTKY3.m2 and PSTI.SSI.in2 Inhibitors

The concentrations ofthe PSTI.OMTKY3 and PSTLSSI inhibitors were determined using the method of Ackermann and Potter [W.W. Ackermann and

V.R. Potter, Proc. Soc. Exp. Biol. Med., 72, 1 (1949)]. Briefly, 8 different concentrations of inhibitor were incubated for one hour at 5 different subtilisn A concentrations in 990μl volumes of assay buffer (lOOmM TRIS, lOmM CaCl₂, 0.05% Triton X-100, pH 8.6). After one hour the 1 l volumes were transferred to cuvettes and the residual subtilisin A activities were measured by addition of lOμl succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanalide [suc-AAPF-pNA; lOOmg/ml in DMSO] (Bache Bioscience, Ine, King of Prussia, PA) The rate of hydrolysis was monitored at 410 nm using a Hewlett-Packard Model 8452A Diode Array Spectrophotometer A subtilisin A control containing no inhibitor was also carried out at each subtilisin A concentration The resultant observed rates of hydrolysis at each inhibitor concentration were plotted against the 5 subtilisin A concentrations The asymptotes to these curves allows for the ready acquisition ofthe inhibitor concentration as the y-intercepts equal -k^I] and the slopes ofthe asymptotes equal k₃ as described by Ackermann and Potter (W W Ackermann and V.R Potter, Proc. Soc. Exp. Biol. Med., 72, 1 (1949)).

Determination of Concentration of PSTI.m2 Inhibitor

The concentration of PSTI. m2 was determined by a modified method of Green and Work (N M. Green and E. Work. Biochem. J. 54, 347, (1953)) A known concentration of trypsin was incubated in assay buffer with various amounts of purified PSTI.m2 for lh at 25°C (50mM TRIS, ImM CaCI₂, pH 8.0). The percent residual activity of trypsin was then measured as described in Example 2, using lOμl BAPNA (Na-benzoyl-DL-arginine p-nitroanilide; 43.5mg/ml) as substrate. The resultant residual activites were then plotted against the volumes of PSTI. m2 inhibitor used in the incubations At a trypsin concentration which is * lOx greater than the K,, there should be a sharp break point in the titration curve. Because this inhibitor forms a l l complex with trypsin, extrapolation of the asymptote from 100% activity down to this break point, such that the asymptote intersects the x-axis was found to be the concentration of inhibitor in units of millimolar

Determination of K, for PSTLSSI

By definition, K, = ^k^,, and as such, independent measurement of both k_off and k_on allowed for ready calculation of K, Determination of l ,_π:

Once having established the concentration ofthe inhibitor, equimolar amounts of subtilisin A and inhibitor were incubated at 25 °C for lh (1 1 complex concentration = 2.5 x IO^"9 M) A 2 5μl aliquot of this 1 1 enzyme "inhibitor complex was removed and added to 997.2μl assay buffer (lOOmM TRIS, lOmM CaCl₂, 0 05% Triton X-100, pH 8 6) containing lOμl suc-AAPF-pNA as substrate (lOOmg/ml in DMSO as stock solution) At the time of addition, the spectrophotometer was started and the rate of hydrolysis of substrate was monitored at 410 nm The rate of hydrolysis was monitored until it reached a constant value corresponding to complete dissociation ofthe complex (maximal hydrolysis rate o the subtilisin A control reaction) The straight line resulting from the fit to maximal rate was extrapolated back to the time axis to obtain the reciprocal (l k_off) ofthe dissociation constant, k_off

Determination of k^:

Using the method described by Tonomura, et al , Interaction of SSI and proteases in solution, Chapter 9 in Protein Protease Inhibitor- The Case of Streptomyces Subtilisin Inhibitor (SSI), Ed Hiromi et al , Elsevier, 1985, the association of subtilisin A with PSTI SSI m2 was followed by measuring the decrease in enzyme activity of subtilisin A and inhibitor in the presence of substrate The association was started by mixing PSTI. SSI. m2 and subtilisin A, and after a certain time interval, an excess amount of substrate was added to the 990μl enzyme.inhibitor solution (final concentrations subtilisin A, 0 268 nM, PSTI SSI m2,4 43 nM, suc-AAPF-pNA, lOμl of lOO g/ml stock in DMSO) Upon addition ofthe substrate, the residual enzyme activity, which is proportional to the free enzyme concentration, was monitored at 410 nm The residual activities were plotted against the time intervals allowed for the association This curve follows the second-order reaction, and the association rate constant, k^, was determined according to the equation shown below

{ 2 303/[Io]-[Eo] } log[Eo]([Io.-[EI]) =

[Io]([Eo]-[EI])

The linear portion of this plot yielded a straight line, with a slope = {[I -[E₀]/2 303 }k_onand a y-mtercept = -log[EJ/[I₀] Therefore, k_on was readily calculated from the slope Results of k_^, k_off, K, determinations are shown in Table I

Table I: Summary of Rate Constants and Dissociation Constants Inhibitor ^ (M-'sec^'1) k^M- ¹) KI M (offfon)

PSTI.m2 3.542 X 10⁵ 0.054 1.524 X I 0^"7

PSTI.OMTKY.m2 1.1012 X 10⁷ 0.0044 4.348 X I 0^"10

PSTI.SSI.m2 1.317 X 10" 0.001 7.593 X IO^'12

Determination of K, for PSTLSSI using IC₅₀ Values

As discussed by Cha (S. Cha Biochem. Pharmacology 24, 2177, (1975)), under experimental conditions outlined above (pre-incubation ofthe inhibitor with the enzyme), the IC₅₀ values one obtains by plotting percent residual activity against [I], are independent of substrate concentration. From Henderson's equations (P.J.F. Henderson, Biochem. J. 127, 321, (1972) it follows that IC₅₀ = 1/2 [E] + K, Therefore, IC₅₀ values were obtained in this manner at 5 (or more) different subtilisin A concentrations. Simple graphical analyses ofthe data were performed: the IC₅₀ values were plotted against enzyme concentration, with the resultant y-intercept equal to K,. K_j values obtained in this manner for all inhibitors showed good agreement with those shown in Table I.

Claims

CLAIMS We claim:

1. A modified subtilisin inhibitor comprising a modified human standard mechanism inhibitor, wherein at least one amino acid ofthe reactive site of said human standard mechanism inhibitor is substituted with a different amino acid, such that the K_j of said modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor.

2. A modified subtilisin inhibitor according to claim 1 wherein at least five amino acids ofthe reactive site of said human standard mechanism inhibitor are substituted with different amino acids.

3. A modified subtilisin inhibitor according to claim 1 wherein the reactive site of said human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor

4 A modified subtilisin inhibitor according to claim 1 wherein the PIO to PIO' residues ofthe reactive site of said human standard mechanism inhibitor are replaced by the PIO to PIO' residues ofthe subtilisin reactive site of a known subtilisin inhibitor

5 A modified subtilisin inhibitor according to claim 1 wherein the P6 to P5' residues of the reactive site of said human standard mechanism inhibitor are replaced by the P6 to P5' residues ofthe subtilisin reactive site of a known subtilisin inhibitor

6 A modified subtilisin inhibitor according to claim 1, 4 or 5 wherein said human standard mechanism inhibitor comprises human pancreatic secretory trypsin inhibitor

7. A modified subtilisin inhibitor according to claim 1 , 4 or 5 wherein said human standard mechanism inhibitor comprises human mucous protease inhibitor.

8. A modified subtilisin inhibitor according to claim 4 or 5 wherein said known subtilisin inhibitor is an avian ovomucoid third domain protein.

9. A modified subtilisin inhibitor according to claim 4 or 5 wherein said known subtilisin inhibitor is a Streptomyces subtilisin inhibitor.

10. A modified subtilisin inhibitor according to claim 9 wherein said avian ovomucoid third domain protein is turkey ovomucoid third domain protein.

1 1. A method of inhibiting a target subtilisin comprising reversibly binding to a target subtilisin a modified subtilisin inhibitor comprising a human standard mechanism inhibitor wherein the reactive site of said human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor and wherein the K, of said modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor.

12. A target subtilisin inhibited by a modified subtilisin inhibitor comprising a human standard mechanism inhibitor wherein the reactive site of said human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor and wherein the K, of said modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor.

13. A nucleic acid encoding a modified human standard mechanism inhibitor, wherein at least one amino acid ofthe reactive site of said human standard mechanism inhibitor is substituted with a different amino acid, such that the K, of said modified subtilisin inhibitor against at least one subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor

14. A nucleic acid according to claim 13 wherein the reactive site of said human standard mechanism inhibitor is replaced by the reactive site of a known subtilisin inhibitor.

15. A nucleic acid according to claim 14 wherein the P6 to P5' residues ofthe reactive site of said human standard mechanism inhibitor are replaced by the P6 to P5' residues of the subtilisin reactivie site of a known subtilisin inhibitor.

16. A nucleic acid according to claim 13 wherein said human standard mechanism inhibitor comprises human pancreatic secretory trypsin inhibitor

17. A nucleic acid according to claim 13 wherein said known subtilisin inhibitor is an avian ovomucoid third domain protein

18. A nucleic acid according to claim 13 wherein said known subtilisin inhibitor is a Streptomyces subtilisin inhibitor.

19. An expression vector comprising the nucleic acid of claim 13

20. A host cell comprising the nucleic acid of claim 13

21 A composition comprising a) a modified subtilisin inhibitor comprising a modified human standard mechanism inhibitor, wherein at least one amino acid ofthe reactive site of said human standard mechanism inhibitor is substituted with a different amino acid, such that the K, of said modified subtilisin inhibitor against a target subtilisin is decreased by at least a factor of 100 relative to the unmodified human standard mechanism inhibitor, and b) a detergent containing a target subtilisin