Mutant proteins having lower allergenic response in humans and methods for constructing, identifying and producing such proteins

Abstract

The present invention relates to a novel improved protein mutant which produces low allergenic response in humans compared to the parent of that mutant. Specifically, the present invention comprises neutralizing or reducing the ability of T-cells to recognize epitopes and thus prevent sensitization of an individual to the protein.

Description

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to proteins which produce lower allergenic response in humans exposed to such proteins, and an assay predictive of such response. More specifically, the present invention relates to a novel improved protein mutant which produces very low allergenic response in humans sensitized to that protein through exposure compared to the precursor of such protein mutant.

B. State of the Art

Proteins used in industrial, pharmaceutical and commercial applications are of increasing prevalence. As a result, the increased exposure due to this prevalence has been responsible for some safety hazards caused by the sensitization of certain persons to those peptides, whereupon subsequent exposure causes extreme allergic reactions which can be injurious and even fatal. For example, proteases are known to cause dangerous hypersensitivity in some individuals. As a result, despite the usefulness of proteases in industry, e.g., in laundry detergents, cosmetics, textile treatment etc. . . . , and the extensive research performed in the field to provide improved proteases which have, for example, more effective stain removal under detergency conditions, the use of proteases in industry has been problematic due to their ability to produce a hypersensitive allergenic response in some humans.

Much work has been done to alleviate these problems. Among the strategies explored to reduce immunogenic potential of protease use have been improved production processes which reduce potential contact by controlling and minimizing workplace concentrations of dust particles or aerosol carrying airborne protease, improved granulation processes which reduce the amount of dust or aerosol actually produced from the protease product, and improved recovery processes to reduce the level of potentially allergenic contaminants in the final product. However, efforts to reduce the allergenicity of protease, per se, have been relatively unsuccessful. Alternatively, efforts have been made to mask epitopes in protease which are recognized by immunoglobulin E (IgE) in hypersensitive individuals (PCT Publication No. WO 92110755) or to enlarge or change the nature of the antigenic determinants by attaching polymers or peptides/proteins to the problematic protease.

When an adaptive immune response occurs in an exaggerated or inappropriate form, the individual experiencing the reaction is said to be hypersensitive. Hypersensitivity reactions are the result of normally beneficial immune responses acting inappropriately and sometimes cause inflammatory reactions and tissue damage. They can be provoked by many antigens; and the cause of a hypersensitivity reaction will vary from one individual to the next. Hypersensitivity does not normally manifest itself upon first contact with the antigen, but usually appears upon subsequent contact. One form of hypersensitivity occurs when an IgE response is directed against innocuous environmental antigens, such as pollen, dust-mites or animal dander. The resulting release of pharmacological mediators by IgE-sensitized mast cells produces an acute inflammatory reaction with symptoms such as asthma or rhinitis.

Nonetheless, a strategy comprising modifying the IgE sites will not generally be successful in preventing the cause of the initial sensitization reaction. Accordingly, such strategies, while perhaps neutralizing or reducing the severity of the subsequent hypersensitivity reaction, will not reduce the number or persons actually sensitized. For example, when a person is known to be hypersensitive to a certain antigen, the general, and only safe, manner of dealing with such a situation is to isolate the hypersensitive person from the antigen as completely as possible. Indeed, any other course of action would be dangerous to the health of the hypersensitive individual. Thus, while reducing the danger of a specific protein for a hypersensitive individual is important, for industrial purposes it would be far more valuable to render a protein incapable of initiating the hypersensitivity reaction in the first place.

T-lymphocytes (T-cells) are key players in the induction and regulation of immune responses and in the execution of immunological effector functions. Specific immunity against infectious agents and tumors is known to be dependent on these cells and they are believed to contribute to the healing of injuries. On the other hand, failure to control these responses can lead to auto aggression. In general, antigen is presented to T-cells in the form of antigen presenting cells which, through a variety of cell surface mechanisms, capture and display antigen or partial antigen in a manner suitable for antigen recognition by the T-cell. Upon recognition of a specific epitope by the receptors on the surface of the T-cells (T-cell receptors), the T-cells begin a series of complex interactions, including proliferation, which result in the production of antibody by B-cells. While T-cells and B-cells are both activated by antigenic epitopes which exist on a given protein or peptide, the actual epitopes recognized by these mononuclear cells are generally not identical. In fact, the epitope which activates a T-cell to initiate the creation of immunologic diversity is quite often not the same epitope which is later recognized by B-cells in the course of the immunologic response. Thus, with respect to hypersensitivity, while the specific antigenic interaction between the T-cell and the antigen is a critical element in the initiation of the immune response to antigenic exposure, the specifics of that interaction, i.e., the epitope recognized, is often not relevant to subsequent development of a full blown allergic reaction.

PCT Publication No. WO 96140791 discloses a process for producing polyalkylene oxide-polypeptide conjugates with reduced allergenicity using polyalkylene oxide as a starting material.

PCT Publication No. WO 97130148 discloses a polypeptide conjugate with reduced allergenicity which comprises one polymeric carrier molecule having two or more polypeptide molecules coupled covalently thereto.

PCT Publication No. WO 96/17929 discloses a process for producing polypeptides with reduced allergenicity comprising the step of conjugating from 1 to 30 polymolecules to a parent polypeptide.

PCT Publication No. WO 92/10755 discloses a method of producing protein variants evoking a reduced immunogenic response in animals. In this application, the proteins of interest, a series of proteases and variants thereof, were used to immunized rats. The sera from the rats was then used to measure the reactivity of the polyclonal antibodies already produced and present in the immunized sera to the protein of interest and variants thereof. From these results, it was possible to determine whether the antibodies in the preparation were comparatively more or less reactive with the protein and its variants, thus permitting an analysis of which changes in the protein are likely to neutralize or reduce the ability of the Ig to bind. From these tests on rats, the conclusion was arrived at that changing any of subtilisin 309 residues corresponding to 127, 128, 129, 130, 131, 151, 136, 151, 152, 153, 154, 161, 162, 163, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 186, 193, 194, 195, 196, 197, 247, 251, 261 will result in a change in the immunological potential.

PCT Publication No. WO 94/10191 discloses low allergenic proteins comprising oligomeric forms of the parent monomeric protein, wherein the oligomer has substantially retained its activity.

The prior art has provided methods of reducing the allergenicity of certain proteins and identification of epitopes which cause allergic reactions in some individuals, the assays used to identify these epitopes generally involving measurement of IgE and IgG antibody in blood sera previously exposed to the antigen. Nonetheless, once an Ig reaction has been initiated, sensitization has already occurred. Accordingly, there is a need for a method of determining epitopes which cause sensitization in the first place, as neutralization of these epitopes will result in significantly less possibility for sensitization to occur, thus reducing the possibility of initial sensitization.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a protein having decreased potential to cause allergenic response in humans compared to a precursor protein.

It is a further object of the present invention to provide for a protease variant which has useful activity in common protease applications, such as detergents and or the treatment of wool to prevent felting, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications such as anti-felting, in cosmetic formulations and for skin care, or as fusion-cleavage enzymes in protein production, which protease variant can be more safely used due to its lowered allergenic potential.

According to the present invention, a method for identifying T-cell epitopes within a protein is provided. The present invention provides an assay which identifies epitopes as follows: antigen presenting cells are combined with naive human T-cells and with a peptide of interest. In a preferred embodiment of the invention, a method is provided wherein a T-cell epitope is recognized comprising the steps of: (a) obtaining from a single blood source a solution of dendritic cells and a solution of naive CD4+ and/or CD8+ T-cells; (b) promoting differentiation in said solution of dendritic cells; (c) combining said solution of differentiated dendritic cells and said naive CD4+ and/or CD8+ T-cells with a peptide of interest; (d) measuring the proliferation of T-cells in said step (c).

According to another embodiment of the present invention, a protein is provided in which a T-cell epitope is modified so as to reduce or preferably neutralize (eliminate) the ability of the T-cell to identify that epitope. Thus, a protein is provided having reduced allergenicity, wherein said protein comprises a modification comprising the substitution or deletion of amino acid residues which are identified as within a T-cell epitope. According to a preferred embodiment, an epitope is determined in a protein or peptide which, when recognized by a T-cell, results in the proliferation of T-cells which is greater than the baseline. That T-cell epitope is then modified so that, when the peptide comprising the epitope is analyzed in the assay of the invention, it results in lesser proliferation than the protein comprising the unmodified epitope. More preferably, the epitope to be modified produces greater than three times the baseline T-cell proliferation in a sample. When modified, the epitope produces less than three times the baseline T-cell proliferation, preferably less than two times the baseline T-cell proliferation and most preferably less than or substantially equal to the baseline T-ell proliferation in a sample.

Preferably, the epitope is modified in one of the following ways: (a) the amino acid sequence of the epitope is substituted with an analogous sequence from a human homolog to the protein of interest, i.e., human subtilisin or another human protease derived subtilisin like molecule such as furin or the kexins (see e.g., Methods in Enzymology, Vol. 244., (1994) pp 175 et seq; Roebroek et al., EMBO J., Vol. 5, No. 9, pp. 2197-2202 (1986); Tomkinson et al., Biochem., Vol. 30, pp. 168-174 (1991); Keifer et al., DNA and Cell Biol., Vol. 10, No. 10, pp. 757-769 (1991)); (b) the amino acid sequence of the epitope is substituted with an analogous sequence from a non-human homolog to the protein of interest, which analogous sequence produces a lesser allergenic response due to T-cell recognition than that of the protein of interest; (c) the amino acid sequence of the epitope is substituted with a sequence which substantially mimics the major tertiary structure attributes of the epitope, but which produces a lesser allergenic response due to T-cell recognition than that of the protein of interest; or (d) with any sequence which produces lesser allergenic response due to T-cell recognition than that of the protein of interest.

In a specific embodiment of the invention, a protease variant is provided comprising at least one amino acid substitution at a position corresponding to residues 170, 171, 172 and/or 173 in BPN′, wherein such substitutions comprise modifying residue 170 to aspartic acid, modifying residue 171 to glutamine, modifying residue 172 to methionine and/or modifying residue 173 to aspartic acid. In a most preferred embodiment, the substitution comprises modifying residues 170, 171 and 173 to aspartic acid, glutamine and aspartic acid, respectively.

In another embodiment of the present invention, a method for producing the protein of the invention having reduced allergenicity is provided. Preferably, the mutant protein is prepared by modifying a DNA encoding a precursor protein so that the modified DNA encodes the mutant protein of the invention.

In yet another embodiment of the invention, DNA sequences encoding the mutant protein, as well as expression vectors containing such DNA sequences and host cells transformed with such vectors are provided, which host cells are preferably capable of expressing such DNA to produce the mutant protein of the invention either intracellularly or extracellulary.

The mutant protein of the invention is useful in any composition or process in which the precursor protein is generally known to be useful. For example, where the protein is a protease, the reduced allergenicity protease can be used as a component in cleaning products such as laundry detergents and hard surface cleansers, as an aid in the preparation of leather, in the treatment of textiles such as wool and/or silk to reduce felting, as a component in a personal care, cosmetic or face cream product, and as a component in animal or pet feed to improve the nutritional value of the feed. Similarly, where the protein is an amylase, the reduce allergenicity amylase can be used for the liquefaction of starch, as a component in a dishwashing detergent, for desizing of textiles, in a laundry detergent or any other use for which amylase is useful.

One advantage of the present invention is that by measuring the proliferation of T-cells due to T-cell epitope recognition, it is possible to identify peptides which contain epitopes responsible for initially sensitizing an individual. That is, T-cell proliferation due to T-cell epitope recognition results in sensitization of an individual to that peptide or a protein which contains it. Neutralization of such “sensitizing” T-cell epitopes will inevitably result in a greater degree of safety for those who handle or are otherwise exposed to the antigen containing the epitope because they will not be initially sensitized, thus preventing the production of Ig antibodies typical of an allergic reaction upon subsequent exposure to the antigen.

An advantage of the present invention is the preparation of proteins, including enzymes, which may be used with significantly less danger of sensitization for the individuals exposed. Thus, for example, the proteins of the invention may be more safely used in cosmetics such as face creams, detergents such as laundry detergents, hard surface cleaning compositions and pre-wash compositions or any other use of protein, including enzymes, wherein human exposure is a necessary by-product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A , B 1 , B 2 and B 3 illustrate the DNA (SEQ ID:NO 1) and amino acid (SEQ ID:NO 2) sequence for Bacillus amyloliquefaciens subtilisin (BPN′) and a partial restriction map of this gene.

FIG. 2 illustrates the conserved amino acid residues among subtilisins from Bacillus amyloliquefaciens (SEQ ID:NO 2) and Bacillus lentus (wild-type) (SEQ ID:NO 5).

FIGS. 3A and 3B illustrate an amino acid sequence alignment of subtilisin type proteases from Bacillus amyloliquefaciens (BPN′) (SEQ ID:NO 2), Bacillus subtilis (SEQ ID:NO 3), Bacillus licheniformis (SEQ ID:NO 4) and Bacillus lentus (SEQ ID:NO 5). The symbol * denotes the absence of specific amino acid residues as compared to subtilisin BPN′.

FIG. 4 illustrates the additive T-cell response of 16 peripheral mononuclear blood samples to peptides corresponding to the Bacillus lentus protease. Peptide E05 includes the region comprising residues corresponding to 170-173 in protease from Bacillus amyloliquefaciens.

FIG. 5 illustrates the additive T-cell response of 10 peripheral mononuclear blood samples to peptides corresponding to the human subtilisin molecule. Peptides F10, F9, F8 and F7 all contain the amino acid sequence DQMD (residues 170-173 of SEQ ID NO; 2) corresponding to the region comprising residues corresponding to 170-173 in protease from Bacillus amyloliquefaciens in the sequence alignment of FIG. 3 .

FIGS. 6 A/ 6 B and FIGS. 6 C/ 6 D/ 6 E illustrate amino acid strings corresponding to peptides derived from the sequence of Bacillus lentus protease and a human subtilisin, respectively.

FIG. 7 illustrates the amino acid sequence of human subtilisin (SEQ ID:NO 7).

FIG. 8 illustrates an amino acid sequence alignment of BPN′ ( Bacillus amyloliquefaciens ) protease (SEQ ID NO: 2), SAVINASE ( Bacillus lentus (SEQ ID NO: 5)) protease and human subtilisin (S2HSBT (SEQ ID NO: 7)).

FIG. 9 illustrates the T-cell response to peptides derived from Bacillus lentus protease in a sample taken from an individual known to be hypersensitive to Bacillus lentus protease. Peptide E05 represents the region corresponding to 170-173 in protease from Bacillus amyloliquefaciens.

FIG. 10 illustrates the T-cell response to various alanine substitutions in the E05 Bacillus lentus protease peptide set in a sample taken from an individual known to be hypersensitive to Bacillus lentus protease.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, a method for identifying T-cell epitopes is provided. The present invention provides an assay which identifies epitopes as follows: differentiated dendritic cells are combined with naive human CD4+ and/or CD8+ T-cells and with a peptide of interest. More specifically, a method is provided wherein a T-cell epitope is recognized comprising the steps of: (a) obtaining from a single blood source a solution of dendritic cells and a solution of naive CD4+ and/or CD8+ T-cells; (b) promoting differentiation in said solution of dendritic cells; (c) combining said solution of differentiated dendritic cells and said naive CD4+ and/or CD8+ T-cells with a peptide of interest; (d) measuring the proliferation of T-cells in said step (c).

The peptide of interest to be analyzed according to the assay of the invention is derived from a protein or enzyme for which reduced allergenicity is desirable or required. In the practice of the invention, it is possible to identify with precision the location of an epitope which can cause sensitization in an individual or sampling of individuals. In a particularly effective embodiment of the invention, a series of peptide oligomers which correspond to all or part of the protein or enzyme are prepared. For example, a peptide library is produced covering the relevant portion or all of the protein. One particularly useful manner of producing the peptides is to introduce overlap into the peptide library, for example, producing a first peptide corresponds to amino acid sequence 1-10 of the subject protein, a second peptide corresponds to amino acid sequence 4-14 of the subject protein, a third peptide corresponds to amino acid sequence 7-17 of the subject protein, a fourth peptide corresponds to amino acid sequence 10-20 of the subject protein etc. . . . until representative peptides corresponding to the entire molecule are created. By analyzing each of the peptides individually in the assay provided herein, it is possible to precisely identify the location of epitopes recognized by T-cells. In the example above, the reaction of one specific peptide to a greater extent than it's neighbors will facilitate identification of the epitope anchor region to within three amino acids. After determining the location of these epitopes, it is possible to alter the amino acids within each epitope until the peptide produces a less significant T-cell response.

“Antigen presenting cell” as used herein means a cell of the immune system which present antigen on their surface which is recognizable by receptors on the surface of T-cells. Examples of antigen presenting cells are dendritic cells, interdigitating cells, activated B-cells and macrophages.

“T-cell proliferation” as used herein means the number of T-cells produced during the incubation of T-cells with the antigen presenting cells, with or without antigen.

“Baseline T-cell proliferation” as used herein means T-cell proliferation which is normally seen in an individual in response to exposure to antigen presenting cells in the absence of peptide or protein antigen. For the purposes herein, the baseline T-cell proliferation level was determined on a per sample basis for each individual as the proliferation of T-cells in response to antigen presenting cells in the absence of antigen.

“T-cell epitope” means a feature of a peptide or protein which is recognized by a T-cell receptor in the initiation of an immunologic response to the peptide comprising that antigen. Recognition of a T-cell epitope by a T-cell is generally believed to be via a mechanism wherein T-cells recognize peptide fragments of antigens which are bound to class I or class II major histocompatability (MHC) molecules expressed on antigen-presenting cells (see e.g., Moeller, G. ed., Antigenic Requirements for Activation of MHC-Restricted Responses, Immunological Review, Vol. 98, p. 187 (Copenhagen; Munksgaard) (1987).

The epitopes determined according to the assay provided herein are then modified to reduce the allergenic potential of the protein of interest. In a preferred embodiment, the epitope to be modified produces a level of T-cell proliferation of greater than three times the baseline T-cell proliferation in a sample. When modified, the epitope produces less than three times the baseline proliferation, preferably less than two times the baseline proliferation and most preferably less than or substantially equal to the baseline proliferation in a sample.

Preferably, the epitope is modified in one of the following ways: (a) the amino acid sequence of the epitope is substituted with an analogous sequence from a human homolog to the protein of interest; (b) the amino acid sequence of the epitope is substituted with an analogous sequence from a non-human homolog to the protein of interest, which analogous sequence produces a lesser allergenic response due to T-cell epitope recognition than that of the protein of interest; (c) the amino acid sequence of the epitope is substituted with a sequence which substantially mimics the major tertiary structure attributes of the epitope, but which produces a lesser allergenic response due to T-cell epitope recognition than that of the protein of interest; or (d) with any sequence which produces lesser allergenic response due to T-cell epitope recognition than that of the protein of interest.

“Sample” as used herein comprises mononuclear cells which are naive, i.e., not sensitized, to the antigen in question.

“Homolog” as used herein means a protein or enzyme which ha s similar catalytic action, structure and/or use as the protein of interest. It is desirable to find a homolog that has a tertiary and/or primary structure similar to the protein of interest as replacement of the epitope in the protein of interest with an analogous segment from the homolog will reduce the disruptiveness of the change. Thus, closely homologous enzymes will provide the most desirable source of epitope substitutions. Alternatively, if possible, it is advantageous to look to human analogs for a given protein. For example, substituting a specific epitope in a bacterial subtilisin with a sequence from a human analog to subtilisin (i.e., human subtilisin) should result in less allergenicity in the bacterial protein.

An “analogous” sequence may be determined by ensuring that the replacement amino acids show a similar function, the tertiary structure and/or conserved residues to the amino acids in the protein of interest at or near the epitope. Thus, where the epitope region contains, for example, an alpha-helix or a beta-sheet structure, the replacement amino acids should maintain that specific structure.

While the present invention extends to all proteins for which it is desired to reduce allergenicity, for the sake of simplicity, the following will describe a particularly preferred embodiment of the invention, the modification of protease. Proteases are carbonyl hydrolases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “protease” means a naturally-occurring protease or a recombinant protease. Naturally-occurring proteases include α-aminoacylpeptide hydrolase, peptidylamino acid hydrolase, acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase, thiol proteinase, carboxylproteinase and metalloproteinase. Seine, metallo, thiol and acid proteases are included, as well as endo and exo-proteases.

Subtilisins are bacterial or fungal proteases which generally act to cleave peptide bonds of proteins or peptides. As used herein, “subtilisin” means a naturally-occurring subtilisin or a recombinant subtilisin. A series of naturally-occurring subtilisins is known to be produced and often secreted by various microbial species. Amino acid sequences of the members of this series are not entirely homologous. However, the subtilisins in this series exhibit the same or similar type of proteolytic activity. This class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. The subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. In the subtilisin related proteases the relative order of these amino acids, reading from the amino to carboxy terminus, is aspartate-histidine-serine. In the chymotrypsin related proteases, the relative order, however, is histidine-aspartate-serine. Thus, subtilisin herein refers to a serine protease having the catalytic triad of subtilisin related proteases. Examples include but are not limited to the subtilisins identified in FIG. 3 herein. Generally and for purposes of the present invention, numbering of the amino acids in proteases corresponds to the numbers assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in FIG. 1 .

“Recombinant subtilisin” or “recombinant protease” refer to a subtilisin or protease in which the DNA sequence encoding the subtilisin or protease is modified to produce a variant (or mutant) DNA sequence which encodes the substitution, deletion or insertion of one or more amino acids in the naturally-occurring amino acid sequence. Suitable methods to produce such modification, and which may be combined with those disclosed herein, include those disclosed in U.S. Pat. No. 4,760,025 (RE 34,606), U.S. Pat. No. 5,204,015 and U.S. Pat. No. 5,185,258.

“Non-human subtilisins” and the DNA encoding them may be obtained from many procaryotic and eucaryotic organisms. Suitable examples of procaryotic organisms include gram negative organisms such as E. coli or Pseudomonas and gram positive bacteria such as Micrococcus or Bacillus. Examples of eucaryotic organisms from which subtilisin and their genes may be obtained include yeast such as Saccharomyces cerevisiae, fungi such as Aspergillus sp.

“Human subtilisin” means proteins of human origin which have subtilisin type catalytic activity, e.g., the kexin family of human derived proteases. An example of such a protein is represented by the sequence in FIG. 7 . Additionally, derivatives or homologs of human subtilisin, including those from non-human sources such as mouse or rabbit, which retain the essential ability to hydrolyze peptide bonds and have at least 50%, preferably at least 65% and most preferably at least 80% homology to the protein of FIG. 7 are considered human subtilisins for the purpose of the invention.

A “protease variant” has an amino add sequence which is derived from the amino acid sequence of a “precursor protease”. The precursor proteases include naturally-occurring proteases and recombinant proteases. The amino acid sequence of the protease variant is “derived” from the precursor protease amino acid sequence by the substitution, deletion or insertion of one or more amino acids of the precursor amino acid sequence. Such modification is of the “precursor DNA sequence” which encodes the amino acid sequence of the precursor protease rather than manipulation of the precursor protease enzyme per se. Suitable methods for such manipulation of the precursor DNA sequence include methods disclosed herein, as well as methods known to those skilled in the art (see, for example, EP 0 328299, W089/06279 and the US patents and applications already referenced herein).

The amino acid position numbers used herein refer to those assigned to the mature Bacillus amyloliquefaciens subtilisin sequence presented in FIG. 1 . The invention, however, is not limited to the mutation of this particular subtilisin but extends to precursor proteases containing amino acid residues at positions which are “equivalent” to the particular identified residues in Bacillus amyloliquefaciens subtilisin. In a preferred embodiment of the present invention, the precursor protease is Bacillus lentus subtilisin and the substitutions, deletions or insertions are made at the equivalent amino acid residue in B. lentus corresponding to those listed above.

A residue (amino acid) of a precursor protease is equivalent to a residue of Bacillus amyloliquefaciens subtilisin if it is either homologous (i.e., corresponding in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in Bacillus amyloliquefaciens subtilisin (i.e., having the same or similar functional capacity to combine, react, or interact chemically).

In order to establish homology to primary structure, the amino acid sequence of a precursor protease is directly compared to the Bacillus amyloliquefaciens subtilisin primary sequence and particularly to a set of residues known to be invariant in subtilisins for which the sequence is known. For example, FIG. 2 herein shows the conserved residues as between B. amyloliquefaciens subtilisin and B. lentus subtilisin. After aligning the conserved residues, allowing for necessary insertions and deletions in order to maintain alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and insertion), the residues equivalent to particular amino acids in the primary sequence of Bacillus amyloliquefaciens subtilisin are defined. Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of greater than 75% or as little as 50% of conserved residues is also adequate to define equivalent residues. Conservation of the catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, the amino acid sequence of subtilisin from Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis (carlsbergensis) and Bacillus lentus can be aligned to provide the maximum amount of homology between amino acid sequences. A comparison of these sequences shows that there are a number of conserved residues contained in each sequence. The conserved residues as between BPN′ and B. lentus are identified in FIG. 2 .

These conserved residues, thus, may be used to define the corresponding equivalent amino acid residues of Bacillus amyloliquefaciens subtilisin in other subtilisins such as subtilisin from Bacillus lentus (PCT Publication No. W089106279 published Jul. 13, 1989), the preferred protease precursor enzyme herein, or the subtilisin referred to as PB92 (EP 0 328 299), which is highly homologous to the preferred Bacillus lentus subtilisin. The amino acid sequences of certain of these subtilisins are aligned in FIGS. 3A and 3B with the sequence of Bacillus amyloliquefaciens subtilisin to produce the maximum homology of conserved residues. As can be seen, there are a number of deletions in the sequence of Bacillus lentus as compared to Bacillus amyloliquefaciens subtilisin. Thus, for example, the equivalent amino acid for Val165 in Bacillus amyloliquefaciens subtilisin in the other subtilisins is isoleucine for B. lentus and B. licheniformis.

Thus, for example, the amino acid at position +170 is lysine (K) in both B. amyloliquefaciens and B. licheniformis subtilisins and arginine (R) in Savinase. In one embodiment of the protease variants of the invention, however, the amino acid equivalent to +170 in Bacillus amyloliquefaciens subtilisin is substituted with aspartic acid (D). The abbreviations and one letter codes for all amino acids in the present invention conform to the Patent In User Manual (GenBank, Mountain View, Calif.) 1990, p.101.

“Equivalent residues” may also be defined by determining homology at the level of tertiary structure for a precursor protease whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the precursor protease and Bacillus amyloliquefaciens subtilisin (N on N, CA on CA, C on C and O on O) are within 0.13nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the protease in question to the Bacillus amyloliquefaciens subtilisin. The best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available. $$ R ⁢   ⁢ factor = ∑ h ⁢ &LeftBracketingBar; Fo ⁡ ( h ) &RightBracketingBar; - &LeftBracketingBar; Fc ⁡ ( h ) &RightBracketingBar; ∑ h ⁢ &LeftBracketingBar; Fo ⁡ ( h ) &RightBracketingBar;

Equivalent residues which are functionally analogous to a specific residue of Bacillus amyloliquefaciens subtilisin are defined as those amino acids of the precursor protease which may adopt a conformation such that they either alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the Bacillus amyloliquefaciens subtilisin. Further, they are those residues of the precursor protease (for which a tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the extent that, although the main chain atoms of the given residue may not satisfy the criteria of equivalence on the basis of occupying a homologous position, the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of Bacillus amyloliquefaciens subtilisin. The coordinates of the three dimensional structure of Bacillus amyloliquefaciens subtilisin are set forth in EPO Publication No. 0 251 446 (equivalent to U.S. Pat. No. 5,182,204, the disclosure of which is incorporated herein by reference) and can be used as outlined above to determine equivalent residues on the level of tertiary structure.

Some of the residues identified for substitution, insertion or deletion are conserved residues whereas others are not. In the case of residues which are not conserved, the replacement of one or more amino acids is limited to substitutions which produce a variant which has an amino acid sequence that does not correspond to one found in nature. In the case of conserved residues, such replacements should not result in a naturally-occurring sequence. The protease variants of the present invention include the mature forms of protease variants, as well as the pro- and prepro- forms of such protease variants. The prepro- forms are the preferred construction since this facilitates the expression, secretion and maturation of the protease variants.

“Prosequence” refers to a sequence of amino acids bound to the N-terminal portion of the mature form of a protease which when removed results in the appearance of the “mature” form of the protease. Many proteolytic enzymes are found in nature as translational proenzyme products and, in the absence of post-translational processing, are expressed in this fashion. A preferred prosequence for producing protease variants is the putative prosequence of Bacillus amyloliquefaciens subtilisin, although other protease prosequences may be used.

A “signal sequence” or “presequence” refers to any sequence of amino acids bound to the N-terminal portion of a protease or to the N-terminal portion of a proprotease which may participate in the secretion of the mature or pro forms of the protease. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protease gene which participate in the effectuation of the secretion of protease under native conditions. The present invention utilizes such sequences to effect the secretion of the protease variants as defined herein. One possible signal sequence comprises the first seven amino acid residues of the signal sequence from Bacillus subtilis subtilisin fused to the remainder of the signal sequence of the subtilisin from Bacillus lentus (ATCC 21536).

A “prepro” form of a protease variant consists of the mature form of the protease having a prosequence operably linked to the amino terminus of the protease and a “pre” or “signal” sequence operably linked to the amino terminus of the prosequence.

“Expression vector” refers to a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of said DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid” and “vector” are sometimes used interchangeably as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which are, or become, known in the art.

The “host cells” used in the present invention generally are procaryotic or eucaryotic hosts which preferably have been manipulated by the methods disclosed in U.S. Pat. No. 4,760,025 (RE 34,606) to render them incapable of secreting enzymatically active endoprotease. A preferred host cell for expressing protease is the Bacillus strain BG2036 which is deficient in enzymatically active neutral protease and alkaline protease (subtilisin). The construction of strain BG2036 is described in detail in U.S. Pat. No. 5,264,366. Other host cells for expressing protease include Bacillus subtilis 1168 (also described in U.S. Pat. No. 4,760,025 (RE 34,606) and U.S. Pat. No. 5,264,366, the disclosure of which are incorporated herein by reference), as well as any suitable Bacillus strain such as B. licheniformis, B. lentus, etc.

Host cells are transformed or transfected with vectors constructed using recombinant DNA techniques. Such transformed host cells are capable of either replicating vectors encoding the protease variants or expressing the desired protease variant. In the case of vectors which encode the pre- or prepro-form of the protease variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.

“Operably linked”, when describing the relationship between two DNA regions, simply means that they are functionally related to each other. For example, a presequence is operably linked to a peptide if it functions as a signal sequence, participating in the secretion of the mature form of the protein most probably involving cleavage of the signal sequence. A promoter is operably linked to a coding sequence if it controls the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.

The genes encoding the naturally-occurring precursor protease may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protease of interest, preparing genomic libraries from organisms expressing the protease, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.

The cloned protease is then used to transform a host cell in order to express the protease. The protease gene is then ligated into a high copy number plasmid. This plasmid replicates in hosts in the sense that it contains the well-known elements necessary for plasmid replication: a promoter operably linked to the gene in question (which may be supplied as the gene's own homologous promoter if it is recognized, i.e., transcribed, by the host), a transcription termination and polyadenylation region (necessary for stability of the mRNA transcribed by the host from the protease gene in certain eucaryotic host cells) which is exogenous or is supplied by the endogenous terminator region of the protease gene and, desirably, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antibiotic-containing media. High copy number plasmids also contain an origin of replication for the host, thereby enabling large numbers of plasmids to be generated in the cytoplasm without chromosomal limitations. However, it is within the scope herein to integrate multiple copies of the protease gene into host genome. This is facilitated by procaryotic and eucaryotic organisms which are particularly susceptible to homologous recombination.

In one embodiment, the gene can be a natural gene such as that from B. lentus or B. amyloliquefaciens. Alternatively, a synthetic gene encoding a naturally-occurring or mutant precursor protease may be produced. In such an approach, the DNA and/or amino acid sequence of the precursor protease is determined. Multiple, overlapping synthetic single-stranded DNA fragments are thereafter synthesized, which upon hybridization and ligation produce a synthetic DNA encoding the precursor protease. An example of synthetic gene construction is set forth in Example 3 of U.S. Pat. No. 5,204,015, the disclosure of which is incorporated herein by reference.

Once the naturally-occurring or synthetic precursor protease gene has been cloned, a number of modifications are undertaken to enhance the use of the gene beyond synthesis of the naturally-occurring precursor protease. Such modifications include the production of recombinant proteases as disclosed in U.S. Pat. No. 4,760,025 (RE 34,606) and EPO Publication No. 0 251 446 and the production of protease variants described herein.

The following cassette mutagenesis method may be used to facilitate the construction of the protease variants of the present invention, although other methods may be used. First, the naturally-occurring gene encoding the protease is obtained and sequenced in whole or in part. Then the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded enzyme. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protease gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the protease gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by M13 primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site.

Once the naturally-occurring DNA or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.

In one aspect of the invention, the objective is to secure a variant protease having altered allergenic potential as compared to the precursor protease, since decreasing such potential enables safer use of the enzyme. While the instant invention is useful to lower allergenic potential, the mutations specified herein may be utilized in combination with mutations known in the art to result altered thermal stability and/or altered substrate specificity, modified activity or altered alkaline stability as compared to the precursor.

Accordingly, the present invention is directed to altering the capability of the T-cell epitope which includes residue positions 170-173 in Bacillus lentus to induce T-cell proliferation. One particularly preferred embodiment of the invention comprises making modification to either one or all of R170D, Y171Q and/or N173D. Similarly, as discussed in detail above, it is believed that the modification of the corresponding residues in any protease will result in a the neutralization of a key T-cell epitope in that protease. Thus, in combination with the presently disclosed mutations in the region corresponding to amino acid residues 170-173, substitutions at positions corresponding to N76D/S103A/V104I/G159D optionally in combination with one or more substitutions selected from the group consisting of positions corresponding to V68A, T213R, A232V, Q236H, Q245R, and T260A of Bacillus amyloliquefaciens subtilisin may be used, in addition to decreasing the allergenic potential of the variant protease of the invention, to modulate overall stability and/or proteolytic activity of the enzyme. Similarly, the substitutions provided herein may be combined with mutation at the Asparagine (N) in Bacillus lentus subtilisin at equivalent position +76 to Aspartate (D) in combination with the mutations S103A/V104I/G159D and optionally in combination with one or more substitutions selected from the group consisting of positions corresponding to V68A, T213R, A232V, Q236H, Q245R, and T260A of Bacillus amyloliquefaciens subtilisin, to produce enhanced stability and/or enhanced activity of the resulting mutant enzyme.

The most preferred embodiments of the invention include the following specific combinations of substituted residues corresponding to positions: N76D/S103A/V104I/G159D/K170D/Y171Q/S173D; V68A/N76D/S103A/V104I/G 159D/K170D/Y171Q/S173D/Q236H; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H/Q245R; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/A232V/Q236H/Q245R; and V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/T213R/A232V/Q236H/Q245R/T260A of Bacillus amyloliquefaciens subtilisin. These substitutions are preferably made in Bacillus lentus (recombinant or native-type) subtilisin, although the substitutions may be made in any Bacillus protease.

Based on the screening results obtained with the variant proteases, the noted mutations noted above in Bacillus amyloliquefaciens subtilisin are important to the proteolytic activity, performance and/or stability of these enzymes and the cleaning or wash performance of such variant enzymes.

Many of the protease variants of the invention are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the protease mutants of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Barry J. Anderson and U.S. Pat. No. 4,261,868 to Jiri Flora, et al. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 (previously incorporated by reference). The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the protease variants of the present invention may be used for any purpose that native or wild-type proteases are used. Thus, these variants can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion-cleavage enzymes in protein production, etc. The variants of the present invention may comprise, in addition to decreased allergenicity, enhanced performance in a detergent composition (as compared to the precursor). As used herein, enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.

Proteases of the invention can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.

The addition of proteases of the invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range, and the temperature is below the described protease's denaturing temperature. In addition, proteases of the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

The variant proteases of the present invention can be included in animal feed such as part of animal feed additives as described in, for example, U.S. Pat. No. 5,612,055; U.S. Pat. No. 5,314,692; and U.S. Pat. No. 5,147,642.

One aspect of the invention is a composition for the treatment of a textile that includes variant proteases of the present invention. The composition can be used to treat for example silk or wool as described in publications such as RD 216,034; EP 134,267; U.S. Pat. No. 4,533,359; and EP 344,259.

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

The variants can be screened for proteolytic activity according to methods well known in the art. Preferred protease variants include multiple substitutions at positions corresponding to: N76D/S103A/V141I/G159D/K170D/Y171Q/S173D; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H/Q245R; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/A232V/Q236H/Q245R; and V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/T213R/A232V/Q236H/Q245 R/T260A of Bacillus amyloliquefaciens subtilisin.

All publications and patents referenced herein are hereby incorporated by reference in their entirety.

EXAMPLES

Example 1

Assay for the Identification of Peptide T-Cell Epitopes Using Naive Human T-Cells

Fresh human peripheral blood cells were collected from “naive” humans, i.e., persons not known to be exposed to or sensitized to Bacillus lentus protease, for determination of antigenic epitopes in protease from Bacillus lentus and human subtilisin. Naive humans is intended to mean that the individual is not known to have been exposed to or developed a reaction to protease in the past. Peripheral mononuclear blood cells (stored at room temperature, no older than 24 hours) were prepared for use as follows: Approximately 30 mis of a solution of buffy coat preparation from one unit of whole blood was brought to 50 ml with Dulbecco's phosphate buffered solution (DPBS) and split into two tubes. The samples were underlaid with 12.5 ml of room temperature lymphoprep density separation media (Nycomed density 1.077 g/ml). The tubes were centrifuged for thirty minutes at 600 G. The interface of the two phases was collected, pooled and washed in DPBS. The cell density of the resultant solution was measured by hemocytometer. Viability was measured by trypan blue exclusion.

From the resulting solution, a differentiated dendritic cell culture was prepared from the peripheral blood mononuclear cell sample having a density of 10 8 cells per 75 ml culture flask in a solution as follows:

(1) 50 ml of serum free AIM V media (Gibco) was supplemented with a 1:100 dilution beta-mercaptoethanol (Gibco). The flasks were laid flat for two hours at 37° C. in 5% CO 2 to allow adherence of monocytes to the flask wall.

(2) Differentiation of the monocyte cells to dendritic cells was as follows: nonadherent cells were removed and the resultant adherent cells (monocytes) combined with 30 ml of AIM V, 800 units/ml of GM-CSF (Endogen) and 500 units/ml of IL4 (Endogen); the resulting mixture was cultured for 5 days under conditions at 37° C. in 5% CO 2 . After five days, the cytokine TNFα (Endogen) was added to 0.2 units/ml, and the cytokine IL-1α (Endogen) was added to a final concentration of 50 units/ml and the mixture incubated at 37° C. in 5% CO 2 for two more days.

(3) On the seventh day, Mitomycin C was added to a concentration of 50 microgram/ml was added to stop growth of the now differentiated dendritic cell culture. The solution was incubated for 60 minutes at 37° C. in 5% CO 2 . Dendritic cells were collected by gently scraping the adherent cells off the bottom of the flask with a cell scraper. Adherent and non-adherent cells were then centrifuged at 600 G for 5 minutes, washed in DPBS and counted.

(4) The prepared dendritic cells were placed into a 96 well round bottom array at 2×10 4 /well in 100 microliter total volume of AIM V media.

CD4+ T cells were prepared from frozen aliquots of the peripheral blood cell samples used to prepare the dendritic cells using the human CD4+ Cellect Kit (Biotex) as per the manufacturers instructions with the following modifications: the aliquots were thawed and washed such that approximately 10 8 cells will be applied per Cellect column; the cells were resuspended in 4 ml DPBS and 1 ml of the Cell reagent from the Cellect Kit, the solution maintained at room temperature for 20 minutes. The resultant solution was centrifuged for five minutes at 600 G at room temperature and the pellet resuspended in 2 ml of DPBS and applied to the Cellect columns. The effluent from the columns was collected in 2% human serum in DPBS. The resultant CD4+ cell solution was centrifuged, resuspended in AIMV media and the density counted.

The CD4+ T-cell suspension was resuspended to a count of 2×10 6 /ml in AIM V media to facilitate efficient manipulation of the 96 well plate.

Peptide antigen is prepared from a 1M stock solution in DMSO by dilution in AIM V media at a 1:10 ratio. 10 microliters of the stock solution is placed in each well of the 96 well plate containing the differentiated dendritic cells. 100 microliter of the diluted CD4+ T-cell solution as prepared above is further added to each well. Useful controls include diluted DMSO blanks, and tetanus toxoid positive controls.

The final concentrations in each well, at 210 microliter total volume are as follows:

2×10 4 CD4+

2×10 5 dendtritic cells (R:S of 10:1)

5 mM peptide

Example 2

Identification of T-Cell Epitopes in Protease from Bacillus lentus and Human subtilisin

Peptides for use in the assay described in Example 1 were prepared based on the Bacillus lentus and human subtilisin amino acid sequence. Peptide antigens were designed as follows. From the full length amino acid sequence of either human subtilisin or Bacillus lentus protease provided in FIG. 1 , 15 mers were synthetically prepared, each 15 mer overlapping with the previous and the subsequent 15 mer except for three residues.

Peptides used correspond to amino acid residue strings in Bacillus lentus as provided in FIG. 8 , and peptides correspond to amino acid residues in human subtilisin as provided in FIG. 7 . The peptides used corresponding to the proteases is provided in FIG. 6 . All tests were performed at least in duplicate. All tests reported displayed robust positive control responses to the antigen tetanus toxoid. Responses were averaged within each experiment, then normalized to the baseline response. A positive event was recorded if the response was at least 3 times the baseline response.

The immunogenic response (i.e., T-cell proliferation) to the prepared peptides from human subtilisin and Bacillus lentus was tallied and is provided in FIGS. 4 and 5 , respectively. T-cell proliferation was measured by the incorporated tritium method. The results shown in FIGS. 4 and 5 as a comparison of the immunogenic additive response in 10 individuals ( FIG. 4 ) and 16 individuals ( FIG. 5 ) to the various peptides. Response is indicated as the added response wherein 1.0 equals a baseline response for each sample. Thus, in FIG. 4 , a reading of 10.0 or less is the baseline response and in FIG. 5 a reading of 16.0 or less the baseline response.

As indicated in FIGS. 4 and 5 , the immunogenic response of the naive blood samples from unsensitized individuals showed a marked allergenic response at the peptide fragment from Bacillus lentus corresponding to residues 170-173 of Bacillus amyloliquefaciens protease. As expected, the corresponding fragment in human subtilisin evokes merely baseline response.

FIG. 9 shows the T-cell response to peptides derived from Bacillus lentus protease in a sample taken from an individual known to be hypersensitive to Bacillus lentus protease. Peptide E05 represents the region corresponding to 170-173 in protease from Bacillus amyloliquefaciens . As shown in FIG. 9 , the hypersensitive individual was highly responsive to the T-cell epitope represented by the peptide E05. This result confirms that, by practicing the assay according to the invention, it is possible to predict the major epitopes identified by the T-cells of a hypersensitive individual.

FIG. 10 shows the T-cell response to various alanine substitutions in the E05 peptide derived from Bacillus lentus protease in a sample taken from an individual known to be hypersensitive to Bacillus lentus protease. Alanine substitutions were used as substitutions for the purpose of determining the role of any specific residue within the epitope. The legend of FIG. 10 refers to the position of the peptide in which an alanine was substituted, i.e., in peptide E06 (sequence GSISYPARYANAMAV) (residues 157-171 at SEQ ID NO: 5), G to A=2, S to A=3, I to A=4, S to A=5,Y to A=6, P to A=7, R to A=8,Y to A=9, N to A=10,M to A=11 and V to A=12. As indicated in FIG. 10 , substitution of either of the residues R170A, Y171A and/or N173A in protease from Bacillus lentus results in dramatically reduced response in the hypersensitive individual's blood sample.

From these results, it is apparent that the residues 170, 171 and 173 are critical for T-cell response within this peptide. Accordingly, it is further apparent that these residues are largely responsible for the initiation of allergic reaction within the protease from Bacillus lentus .

#             SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS:  7
<210> SEQ ID NO 1
<211> LENGTH: 1494
<212> TYPE: DNA
<213> ORGANISM: Bacillus amyloliquefaciens
<220> FEATURE:
<221> NAME/KEY: CDS
<222> LOCATION: (96)..(1244)
<221> NAME/KEY: mat_peptide
<222> LOCATION: (417)..(1241)
<400> SEQUENCE: 1
ggtctactaa aatattattc catactatac aattaataca cagaataatc tg
#tctattgg     60
ttattctgca aatgaaaaaa aggagaggat aaaga gtg aga ggc
#aaa aaa gta       113
#
#   Val Arg Gly Lys Lys Val
#
#          -105
tgg atc agt ttg ctg ttt gct tta gcg tta at
#c ttt acg atg gcg ttc      161
Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu Il
#e Phe Thr Met Ala Phe
-100
#   -95
#   -90
ggc agc aca tcc tct gcc cag gcg gca ggg aa
#a tca aac ggg gaa aag      209
Gly Ser Thr Ser Ser Ala Gln Ala Ala Gly Ly
#s Ser Asn Gly Glu Lys
-85                 -
#80                 -
#75                 -
#70
aaa tat att gtc ggg ttt aaa cag aca atg ag
#c acg atg agc gcc gct      257
Lys Tyr Ile Val Gly Phe Lys Gln Thr Met Se
#r Thr Met Ser Ala Ala
-65
#               -60
#               -55
aag aag aaa gat gtc att tct gaa aaa ggc gg
#g aaa gtg caa aag caa      305
Lys Lys Lys Asp Val Ile Ser Glu Lys Gly Gl
#y Lys Val Gln Lys Gln
-50
#           -45
#           -40
ttc aaa tat gta gac gca gct tca gct aca tt
#a aac gaa aaa gct gta      353
Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr Le
#u Asn Glu Lys Ala Val
-35
#       -30
#       -25
aaa gaa ttg aaa aaa gac ccg agc gtc gct ta
#c gtt gaa gaa gat cac      401
Lys Glu Leu Lys Lys Asp Pro Ser Val Ala Ty
#r Val Glu Glu Asp His
-20
#   -15
#   -10
gta gca cat gcg tac gcg cag tcc gtg cct ta
#c ggc gta tca caa att      449
Val Ala His Ala Tyr Ala Gln Ser Val Pro Ty
#r Gly Val Ser Gln Ile
-5              -1
# 1               5
#                10
aaa gcc cct gct ctg cac tct caa ggc tac ac
#t gga tca aat gtt aaa      497
Lys Ala Pro Ala Leu His Ser Gln Gly Tyr Th
#r Gly Ser Asn Val Lys
15
#             20
#             25
gta gcg gtt atc gac agc ggt atc gat tct tc
#t cat cct gat tta aag      545
Val Ala Val Ile Asp Ser Gly Ile Asp Ser Se
#r His Pro Asp Leu Lys
30
#         35
#         40
gta gca ggc gga gcc agc atg gtt cct tct ga
#a aca aat cct ttc caa      593
Val Ala Gly Gly Ala Ser Met Val Pro Ser Gl
#u Thr Asn Pro Phe Gln
45
#     50
#     55
gac aac aac tct cac gga act cac gtt gcc gg
#c aca gtt gcg gct ctt      641
Asp Asn Asn Ser His Gly Thr His Val Ala Gl
#y Thr Val Ala Ala Leu
60
# 65
# 70
# 75
aat aac tca atc ggt gta tta ggc gtt gcg cc
#a agc gca tca ctt tac      689
Asn Asn Ser Ile Gly Val Leu Gly Val Ala Pr
#o Ser Ala Ser Leu Tyr
80
#                 85
#                 90
gct gta aaa gtt ctc ggt gct gac ggt tcc gg
#c caa tac agc tgg atc      737
Ala Val Lys Val Leu Gly Ala Asp Gly Ser Gl
#y Gln Tyr Ser Trp Ile
95
#            100
#            105
att aac gga atc gag tgg gcg atc gca aac aa
#t atg gac gtt att aac      785
Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn As
#n Met Asp Val Ile Asn
110
#       115
#       120
atg agc ctc ggc gga cct tct ggt tct gct gc
#t tta aaa gcg gca gtt      833
Met Ser Leu Gly Gly Pro Ser Gly Ser Ala Al
#a Leu Lys Ala Ala Val
125
#   130
#   135
gat aaa gcc gtt gca tcc ggc gtc gta gtc gt
#t gcg gca gcc ggt aac      881
Asp Lys Ala Val Ala Ser Gly Val Val Val Va
#l Ala Ala Ala Gly Asn
140                 1
#45                 1
#50                 1
#55
gaa ggc act tcc ggc agc tca agc aca gtg gg
#c tac cct ggt aaa tac      929
Glu Gly Thr Ser Gly Ser Ser Ser Thr Val Gl
#y Tyr Pro Gly Lys Tyr
160
#               165
#               170
cct tct gtc att gca gta ggc gct gtt gac ag
#c agc aac caa aga gca      977
Pro Ser Val Ile Ala Val Gly Ala Val Asp Se
#r Ser Asn Gln Arg Ala
175
#           180
#           185
tct ttc tca agc gta gga cct gag ctt gat gt
#c atg gca cct ggc gta     1025
Ser Phe Ser Ser Val Gly Pro Glu Leu Asp Va
#l Met Ala Pro Gly Val
190
#       195
#       200
tct atc caa agc acg ctt cct gga aac aaa ta
#c ggg gcg tac aac ggt     1073
Ser Ile Gln Ser Thr Leu Pro Gly Asn Lys Ty
#r Gly Ala Tyr Asn Gly
205
#   210
#   215
acg tca atg gca tct ccg cac gtt gcc gga gc
#g gct gct ttg att ctt     1121
Thr Ser Met Ala Ser Pro His Val Ala Gly Al
#a Ala Ala Leu Ile Leu
220                 2
#25                 2
#30                 2
#35
tct aag cac ccg aac tgg aca aac act caa gt
#c cgc agc agt tta gaa     1169
Ser Lys His Pro Asn Trp Thr Asn Thr Gln Va
#l Arg Ser Ser Leu Glu
240
#               245
#               250
aac acc act aca aaa ctt ggt gat tct ttc ta
#c tat gga aaa ggg ctg     1217
Asn Thr Thr Thr Lys Leu Gly Asp Ser Phe Ty
#r Tyr Gly Lys Gly Leu
255
#           260
#           265
atc aac gta cag gcg gca gct cag taa aacataaaa
#a accggccttg           1264
Ile Asn Val Gln Ala Ala Ala Gln
270
#       275
gccccgccgg ttttttattt ttcttcctcc gcatgttcaa tccgctccat aa
#tcgacgga   1324
tggctccctc tgaaaatttt aacgagaaac ggcgggttga cccggctcag tc
#ccgtaacg   1384
gccaagtcct gaaacgtctc aatcgccgct tcccggtttc cggtcagctc aa
#tgccgtaa   1444
cggtcggcgg cgttttcctg ataccgggag acggcattcg taatcggatc
#            1494
<210> SEQ ID NO 2
<211> LENGTH: 382
<212> TYPE: PRT
<213> ORGANISM: Bacillus amyloliquefaciens
<400> SEQUENCE: 2
Val Arg Gly Lys Lys Val Trp Ile Ser Leu Le
#u Phe Ala Leu Ala Leu
1               5
#                 10
#                 15
Ile Phe Thr Met Ala Phe Gly Ser Thr Ser Se
#r Ala Gln Ala Ala Gly
20
#             25
#             30
Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gl
#y Phe Lys Gln Thr Met
35
#         40
#         45
Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Va
#l Ile Ser Glu Lys Gly
50
#     55
#     60
Gly Lys Val Gln Lys Gln Phe Lys Tyr Val As
#p Ala Ala Ser Ala Thr
65
# 70
# 75
# 80
Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Ly
#s Asp Pro Ser Val Ala
85
#                 90
#                 95
Tyr Val Glu Glu Asp His Val Ala His Ala Ty
#r Ala Gln Ser Val Pro
100
#           105
#           110
Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Le
#u His Ser Gln Gly Tyr
115
#       120
#       125
Thr Gly Ser Asn Val Lys Val Ala Val Ile As
#p Ser Gly Ile Asp Ser
130
#   135
#   140
Ser His Pro Asp Leu Lys Val Ala Gly Gly Al
#a Ser Met Val Pro Ser
145                 1
#50                 1
#55                 1
#60
Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser Hi
#s Gly Thr His Val Ala
165
#               170
#               175
Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gl
#y Val Leu Gly Val Ala
180
#           185
#           190
Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Le
#u Gly Ala Asp Gly Ser
195
#       200
#       205
Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Gl
#u Trp Ala Ile Ala Asn
210
#   215
#   220
Asn Met Asp Val Ile Asn Met Ser Leu Gly Gl
#y Pro Ser Gly Ser Ala
225                 2
#30                 2
#35                 2
#40
Ala Leu Lys Ala Ala Val Asp Lys Ala Val Al
#a Ser Gly Val Val Val
245
#               250
#               255
Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gl
#y Ser Ser Ser Thr Val
260
#           265
#           270
Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Al
#a Val Gly Ala Val Asp
275
#       280
#       285
Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Va
#l Gly Pro Glu Leu Asp
290
#   295
#   300
Val Met Ala Pro Gly Val Ser Ile Gln Ser Th
#r Leu Pro Gly Asn Lys
305                 3
#10                 3
#15                 3
#20
Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Se
#r Pro His Val Ala Gly
325
#               330
#               335
Ala Ala Ala Leu Ile Leu Ser Lys His Pro As
#n Trp Thr Asn Thr Gln
340
#           345
#           350
Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Ly
#s Leu Gly Asp Ser Phe
355
#       360
#       365
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Al
#a Ala Ala Gln
370
#   375
#   380
<210> SEQ ID NO 3
<211> LENGTH: 275
<212> TYPE: PRT
<213> ORGANISM: Bacillus subtilis
<400> SEQUENCE: 3
Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Il
#e Lys Ala Pro Ala Leu
1               5
#                 10
#                 15
His Ser Gln Gly Tyr Thr Gly Ser Asn Val Ly
#s Val Ala Val Ile Asp
20
#             25
#             30
Ser Gly Ile Asp Ser Ser His Pro Asp Leu As
#n Val Arg Gly Gly Ala
35
#         40
#         45
Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr Gl
#n Asp Gly Ser Ser His
50
#     55
#     60
Gly Thr His Val Ala Gly Thr Ile Ala Ala Le
#u Asn Asn Ser Ile Gly
65
# 70
# 75
# 80
Val Leu Gly Val Ser Pro Ser Ala Ser Leu Ty
#r Ala Val Lys Val Leu
85
#                 90
#                 95
Asp Ser Thr Gly Ser Gly Gln Tyr Ser Trp Il
#e Ile Asn Gly Ile Glu
100
#           105
#           110
Trp Ala Ile Ser Asn Asn Met Asp Val Ile As
#n Met Ser Leu Gly Gly
115
#       120
#       125
Pro Thr Gly Ser Thr Ala Leu Lys Thr Val Va
#l Asp Lys Ala Val Ser
130
#   135
#   140
Ser Gly Ile Val Val Ala Ala Ala Ala Gly As
#n Glu Gly Ser Ser Gly
145                 1
#50                 1
#55                 1
#60
Ser Thr Ser Thr Val Gly Tyr Pro Ala Lys Ty
#r Pro Ser Thr Ile Ala
165
#               170
#               175
Val Gly Ala Val Asn Ser Ser Asn Gln Arg Al
#a Ser Phe Ser Ser Ala
180
#           185
#           190
Gly Ser Glu Leu Asp Val Met Ala Pro Gly Va
#l Ser Ile Gln Ser Thr
195
#       200
#       205
Leu Pro Gly Gly Thr Tyr Gly Ala Tyr Asn Gl
#y Thr Ser Met Ala Thr
210
#   215
#   220
Pro His Val Ala Gly Ala Ala Ala Leu Ile Le
#u Ser Lys His Pro Thr
225                 2
#30                 2
#35                 2
#40
Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Gl
#u Ser Thr Ala Thr Tyr
245
#               250
#               255
Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Le
#u Ile Asn Val Gln Ala
260
#           265
#           270
Ala Ala Gln
275
<210> SEQ ID NO 4
<211> LENGTH: 274
<212> TYPE: PRT
<213> ORGANISM: Bacillus licheniformis
<400> SEQUENCE: 4
Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Il
#e Lys Ala Asp Lys Val
1               5
#                 10
#                 15
Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Ly
#s Val Ala Val Leu Asp
20
#             25
#             30
Thr Gly Ile Gln Ala Ser His Pro Asp Leu As
#n Val Val Gly Gly Ala
35
#         40
#         45
Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr As
#p Gly Asn Gly His Gly
50
#     55
#     60
Thr His Val Ala Gly Thr Val Ala Ala Leu As
#p Asn Thr Thr Gly Val
65
# 70
# 75
# 80
Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Al
#a Val Lys Val Leu Asn
85
#                 90
#                 95
Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Va
#l Ser Gly Ile Glu Trp
100
#           105
#           110
Ala Thr Thr Asn Gly Met Asp Val Ile Asn Me
#t Ser Leu Gly Gly Ala
115
#       120
#       125
Ser Gly Ser Thr Ala Met Lys Gln Ala Val As
#p Asn Ala Tyr Ala Arg
130
#   135
#   140
Gly Val Val Val Val Ala Ala Ala Gly Asn Se
#r Gly Asn Ser Gly Ser
145                 1
#50                 1
#55                 1
#60
Thr Asn Thr Ile Gly Tyr Pro Ala Lys Tyr As
#p Ser Val Ile Ala Val
165
#               170
#               175
Gly Ala Val Asp Ser Asn Ser Asn Arg Ala Se
#r Phe Ser Ser Val Gly
180
#           185
#           190
Ala Glu Leu Glu Val Met Ala Pro Gly Ala Gl
#y Val Tyr Ser Thr Tyr
195
#       200
#       205
Pro Thr Asn Thr Tyr Ala Thr Leu Asn Gly Th
#r Ser Met Ala Ser Pro
210
#   215
#   220
His Val Ala Gly Ala Ala Ala Leu Ile Leu Se
#r Lys His Pro Asn Leu
225                 2
#30                 2
#35                 2
#40
Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Se
#r Thr Ala Thr Tyr Leu
245
#               250
#               255
Gly Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Il
#e Asn Val Glu Ala Ala
260
#           265
#           270
Ala Gln
<210> SEQ ID NO 5
<211> LENGTH: 269
<212> TYPE: PRT
<213> ORGANISM: Bacillus lentus
<400> SEQUENCE: 5
Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Va
#l Gln Ala Pro Ala Ala
1               5
#                 10
#                 15
His Asn Arg Gly Leu Thr Gly Ser Gly Val Ly
#s Val Ala Val Leu Asp
20
#             25
#             30
Thr Gly Ile Ser Thr His Pro Asp Leu Asn Il
#e Arg Gly Gly Ala Ser
35
#         40
#         45
Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gl
#y Asn Gly His Gly Thr
50
#     55
#     60
His Val Ala Gly Thr Ile Ala Ala Leu Asn As
#n Ser Ile Gly Val Leu
65
# 70
# 75
# 80
Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Va
#l Lys Val Leu Gly Ala
85
#                 90
#                 95
Ser Gly Ser Gly Ser Val Ser Ser Ile Ala Gl
#n Gly Leu Glu Trp Ala
100
#           105
#           110
Gly Asn Asn Gly Met His Val Ala Asn Leu Se
#r Leu Gly Ser Pro Ser
115
#       120
#       125
Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Se
#r Ala Thr Ser Arg Gly
130
#   135
#   140
Val Leu Val Val Ala Ala Ser Gly Asn Ser Gl
#y Ala Gly Ser Ile Ser
145                 1
#50                 1
#55                 1
#60
Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Va
#l Gly Ala Thr Asp Gln
165
#               170
#               175
Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gl
#y Ala Gly Leu Asp Ile
180
#           185
#           190
Val Ala Pro Gly Val Asn Val Gln Ser Thr Ty
#r Pro Gly Ser Thr Tyr
195
#       200
#       205
Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pr
#o His Val Ala Gly Ala
210
#   215
#   220
Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Tr
#p Ser Asn Val Gln Ile
225                 2
#30                 2
#35                 2
#40
Arg Asn His Leu Lys Asn Thr Ala Thr Ser Le
#u Gly Ser Thr Asn Leu
245
#               250
#               255
Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Al
#a Thr Arg
260
#           265
<210> SEQ ID NO 6
<211> LENGTH: 15
<212> TYPE: PRT
<213> ORGANISM: Unknown
<220> FEATURE:
<223> OTHER INFORMATION: Description of Unknown Or
#ganism: Unknown
Sequence
<400> SEQUENCE: 6
Ile Lys Asp Phe His Val Tyr Phe Arg Glu Se
#r Arg Asp Ala Gly
1               5
#                 10
#                 15
<210> SEQ ID NO 7
<211> LENGTH: 1052
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 7
Met Lys Leu Val Asn Ile Trp Leu Leu Leu Le
#u Val Val Leu Leu Cys
1               5
#                 10
#                 15
Gly Lys Lys His Leu Gly Asp Arg Leu Glu Ly
#s Lys Ser Phe Glu Lys
20
#             25
#             30
Ala Pro Cys Pro Gly Cys Ser His Leu Thr Le
#u Lys Val Glu Phe Ser
35
#         40
#         45
Ser Thr Val Val Glu Tyr Glu Tyr Ile Val Al
#a Phe Asn Gly Tyr Phe
50
#     55
#     60
Thr Ala Lys Ala Arg Asn Ser Phe Ile Ser Se
#r Ala Leu Lys Ser Ser
65
# 70
# 75
# 80
Glu Val Asp Asn Trp Arg Ile Ile Pro Arg As
#n Asn Pro Ser Ser Asp
85
#                 90
#                 95
Tyr Pro Ser Asp Phe Glu Val Ile Gln Ile Ly
#s Glu Lys Gln Lys Ala
100
#           105
#           110
Gly Leu Leu Thr Leu Glu Asp His Pro Asn Il
#e Lys Arg Val Thr Pro
115
#       120
#       125
Gln Arg Lys Val Phe Arg Ser Leu Lys Tyr Al
#a Glu Ser Asp Pro Thr
130
#   135
#   140
Val Pro Cys Asn Glu Thr Arg Trp Ser Gln Ly
#s Trp Gln Ser Ser Arg
145                 1
#50                 1
#55                 1
#60
Pro Leu Arg Arg Ala Ser Leu Ser Leu Gly Se
#r Gly Phe Trp His Ala
165
#               170
#               175
Thr Gly Arg His Ser Ser Arg Arg Leu Leu Ar
#g Ala Ile Pro Arg Gln
180
#           185
#           190
Val Ala Gln Thr Leu Gln Ala Asp Val Leu Tr
#p Gln Met Gly Tyr Thr
195
#       200
#       205
Gly Ala Asn Val Arg Val Ala Val Phe Asp Th
#r Gly Leu Ser Glu Lys
210
#   215
#   220
His Pro His Phe Lys Asn Val Lys Glu Arg Th
#r Asn Trp Thr Asn Glu
225                 2
#30                 2
#35                 2
#40
Arg Thr Leu Asp Asp Gly Leu Gly His Gly Th
#r Phe Val Ala Gly Val
245
#               250
#               255
Ile Ala Ser Met Arg Glu Cys Gln Gly Phe Al
#a Pro Asp Ala Glu Leu
260
#           265
#           270
His Ile Phe Arg Val Phe Thr Asn Asn Gln Va
#l Ser Tyr Thr Ser Trp
275
#       280
#       285
Phe Leu Asp Ala Phe Asn Tyr Ala Ile Leu Ly
#s Lys Ile Asp Val Leu
290
#   295
#   300
Asn Leu Ser Ile Gly Gly Pro Asp Phe Met As
#p His Pro Phe Val Asp
305                 3
#10                 3
#15                 3
#20
Lys Val Trp Glu Leu Thr Ala Asn Asn Val Il
#e Met Val Ser Ala Ile
325
#               330
#               335
Gly Asn Asp Gly Pro Leu Tyr Gly Thr Leu As
#n Asn Pro Ala Asp Gln
340
#           345
#           350
Met Asp Val Ile Gly Val Gly Gly Ile Asp Ph
#e Glu Asp Asn Ile Ala
355
#       360
#       365
Arg Phe Ser Ser Arg Gly Met Thr Thr Trp Gl
#u Leu Pro Gly Gly Tyr
370
#   375
#   380
Gly Arg Met Lys Pro Asp Ile Val Thr Tyr Gl
#y Ala Gly Val Arg Gly
385                 3
#90                 3
#95                 4
#00
Ser Gly Val Lys Gly Gly Cys Arg Ala Leu Se
#r Gly Thr Ser Val Ala
405
#               410
#               415
Ser Pro Val Val Ala Gly Ala Val Thr Leu Le
#u Val Ser Thr Val Gln
420
#           425
#           430
Lys Arg Glu Leu Val Asn Pro Ala Ser Met Ly
#s Gln Ala Leu Ile Ala
435
#       440
#       445
Ser Ala Arg Arg Leu Pro Gly Val Asn Met Ph
#e Glu Gln Gly His Gly
450
#   455
#   460
Lys Leu Asp Leu Leu Arg Ala Tyr Gln Ile Le
#u Asn Ser Tyr Lys Pro
465                 4
#70                 4
#75                 4
#80
Gln Ala Ser Leu Ser Pro Ser Tyr Ile Asp Le
#u Thr Glu Cys Pro Tyr
485
#               490
#               495
Met Trp Pro Tyr Cys Ser Gln Pro Ile Tyr Ty
#r Gly Gly Met Pro Thr
500
#           505
#           510
Val Val Asn Val Thr Ile Leu Asn Gly Met Gl
#y Val Thr Gly Arg Ile
515
#       520
#       525
Val Asp Lys Pro Asp Trp Gln Pro Tyr Leu Pr
#o Gln Asn Gly Asp Asn
530
#   535
#   540
Ile Glu Val Ala Phe Ser Tyr Ser Ser Val Le
#u Trp Pro Trp Ser Gly
545                 5
#50                 5
#55                 5
#60
Tyr Leu Ala Ile Ser Ile Ser Val Thr Lys Ly
#s Ala Ala Ser Trp Glu
565
#               570
#               575
Gly Ile Ala Gln Gly His Val Met Ile Thr Va
#l Ala Ser Pro Ala Glu
580
#           585
#           590
Thr Glu Ser Lys Asn Gly Ala Glu Gln Thr Se
#r Thr Val Lys Leu Pro
595
#       600
#       605
Ile Lys Val Lys Ile Ile Pro Thr Pro Pro Ar
#g Ser Lys Arg Val Leu
610
#   615
#   620
Trp Asp Gln Tyr His Asn Leu Arg Tyr Pro Pr
#o Gly Tyr Phe Pro Arg
625                 6
#30                 6
#35                 6
#40
Asp Asn Leu Arg Met Lys Asn Asp Pro Leu As
#p Trp Asn Gly Asp His
645
#               650
#               655
Ile His Thr Asn Phe Arg Asp Met Tyr Gln Hi
#s Leu Arg Ser Met Gly
660
#           665
#           670
Tyr Phe Val Glu Val Leu Gly Ala Pro Phe Th
#r Cys Phe Asp Ala Ser
675
#       680
#       685
Gln Tyr Gly Thr Leu Leu Met Val Asp Ser Gl
#u Glu Glu Tyr Phe Pro
690
#   695
#   700
Glu Glu Ile Ala Lys Leu Arg Arg Asp Val As
#p Asn Gly Leu Ser Leu
705                 7
#10                 7
#15                 7
#20
Val Ile Phe Ser Asp Trp Tyr Asn Thr Ser Va
#l Met Arg Lys Val Lys
725
#               730
#               735
Phe Tyr Asp Glu Asn Thr Arg Gln Trp Trp Me
#t Pro Asp Thr Gly Gly
740
#           745
#           750
Ala Asn Ile Pro Ala Leu Asn Glu Leu Leu Se
#r Val Trp Asn Met Gly
755
#       760
#       765
Phe Ser Asp Gly Leu Tyr Glu Gly Glu Phe Th
#r Leu Ala Asn His Asp
770
#   775
#   780
Met Tyr Tyr Ala Ser Gly Cys Ser Ile Ala Ly
#s Phe Pro Glu Asp Gly
785                 7
#90                 7
#95                 8
#00
Val Val Ile Thr Gln Thr Phe Lys Asp Gln Gl
#y Leu Glu Val Leu Lys
805
#               810
#               815
Gln Glu Thr Ala Val Val Glu Asn Val Pro Il
#e Leu Gly Leu Tyr Gln
820
#           825
#           830
Ile Pro Ala Glu Gly Gly Gly Arg Ile Val Le
#u Tyr Gly Asp Ser Asn
835
#       840
#       845
Cys Leu Asp Asp Ser His Arg Gln Lys Asp Cy
#s Phe Trp Leu Leu Asp
850
#   855
#   860
Ala Leu Leu Gln Tyr Thr Ser Tyr Gly Val Th
#r Pro Pro Ser Leu Ser
865                 8
#70                 8
#75                 8
#80
His Ser Gly Asn Arg Gln Arg Pro Pro Ser Gl
#y Ala Gly Ser Val Thr
885
#               890
#               895
Pro Glu Arg Met Glu Gly Asn His Leu His Ar
#g Tyr Ser Lys Val Leu
900
#           905
#           910
Glu Ala His Leu Gly Asp Pro Lys Pro Arg Pr
#o Leu Pro Ala Cys Pro
915
#       920
#       925
Arg Leu Ser Trp Ala Lys Pro Gln Pro Leu As
#n Glu Thr Ala Pro Ser
930
#   935
#   940
Asn Leu Trp Lys His Gln Lys Leu Leu Ser Il
#e Asp Leu Asp Lys Val
945                 9
#50                 9
#55                 9
#60
Val Leu Pro Asn Phe Arg Ser Asn Arg Pro Gl
#n Val Arg Pro Leu Ser
965
#               970
#               975
Pro Gly Glu Ser Gly Ala Trp Asp Ile Pro Gl
#y Gly Ile Met Pro Gly
980
#           985
#           990
Arg Tyr Asn Gln Glu Val Gly Gln Thr Ile Pr
#o Val Phe Ala Phe Leu
995
#       1000
#      1005
Gly Ala Met Val Val Leu Ala Phe Phe Val Va
#l Gln Ile Asn Lys Ala
1010
#   1015
#  1020
Lys Ser Arg Pro Lys Arg Arg Lys Pro Arg Va
#l Lys Arg Pro Gln Leu
1025                1030
#                1035
#               1040
Met Gln Gln Val His Pro Pro Lys Thr Pro Se
#r Val
1045
#               1050

Claims

1. A protease variant comprising a substitution made at Y171Q and at one or both of the positions in a precursor protease corresponding to K170D and/or S173D of Bacillus amyloliquefaciens subtilisin.

2. A protease variant according to claim 1, comprising combined substitution sets selected from the group consisting of positions corresponding to K170D/Y171Q/S173D; N76D/S103A/V104I/G159D/K170D/Y171Q/S173D; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/Q236H/Q245R; V68A/N76D/S103A/V104I/G159D/K170D/Y171Q/S173D/A232V/Q236H/Q245R; and V68A/N76D/S103A/V104I/G159D/K170D/Y171S/S173D/T213R/A232V/Q236H/Q245R/T260A wherein K170D/Y171Q/S173D positions are found in Bacilus amyloliquefaciens subtilisin and the remaining positions are found in Bacillus lentis subtilisin.

3. A protease variant comprising a substitution made at one or both of the positions in a precursor protease corresponding to K170D and/or S173D of Bacillus amyloliquefaciens subtilisin.

4. A protease variant according to claim 3, comprising combined substitution sets selected from the group consisting of positions corresponding to K170D/S173D;N76D/S103A/V104I/G159D/K170D/S173D; V68A/N76D/S103A/V104I/G159D/K170D/S173D/Q236H; V68A/N76D/S103A/V104I/G159D/K170D/S173D/Q236H/Q245R; V68A/N76D/S103A/V104I/G159D/K170D/S173D/A232V/Q236H/Q245R; and V68A/N76D/S103A/V104I/G159D/K170D/S173D/T213R/A232V/Q236H/Q245R/T260A; wherein K170D/S173D positions are found in Bacilus amyloliquefaciens subtilisin and the remaining positions are found in Bacillus lentis subtilisin.

5. The protease variant according to claim 1 or 3, further comprising a substitution at one or more positions in a precursor protease equivalent to those selected from the group consisting of N76D, S103A, V104I, G159D, V68A, T213R, A232V, Q236H, Q245R, and T260A of Bacillus lentis subtilisin.

6. The protease variant according to claim 5 which is derived from a Bacillus subtilisin.

7. The protease variant according to claim 6 which is derived from Bacillus lentus subtilisin or Bacillus amyloliquefaciens subtilisin.

8. A cleaning composition comprising the protease variant of claim 1 or 3.

9. An animal feed composition comprising the protease variant of claim 1 or 3.

10. A composition comprising the protease variant of claim 1 or 3 for treating a textile.