JP2001510050A - Proteases from Gram-positive microorganisms - Google Patents

Abstract

  (57) [Summary] The present invention relates to the identification of new cysteine proteases in Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences of Bacillus subtilis cysteine proteases CP1, CP2 and CP3. The invention also provides a host cell in which some or all of the genes encoding CP1, CP2 or CP3 have been mutated or deleted. The invention also provides a host cell comprising a nucleic acid encoding a desired heterologous protein, such as an enzyme. The present invention also provides a cleaning composition comprising the cysteine protease of the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] TECHNICAL FIELD The present invention to which the invention pertains, the present invention relates to a cysteine protease from Gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences of cysteine proteases 1, 2 and 3 identified in Bacillus. The present invention also provides a method for producing cysteine proteases 1, 2 and 3 in a host cell, and a variant in a host cell in which at least some or all of the cysteine proteases of the invention have been mutated or deleted. A method for producing a protein is provided.

[0002] The onset Akira of Gram-positive microorganisms such as background bacteria of the genus Bacillus, in part, because of their ability to secrete into the culture medium during fermentation-producing organisms, have been used for large-scale industrial fermentation Was. In Gram-positive bacteria, secreted proteins are transported across cell membranes and cell walls and subsequently released into external media, which usually maintain their innate conformation.

[0003] Various Gram-positive microorganisms are known to secrete extracellular and / or intracellular proteases at several stages in their life cycle. Many proteases are produced in large quantities for industrial purposes. A negative aspect of the presence of proteases in Gram-positive microorganisms is that they contribute to the overall degradation of secreted variants or heterologous proteins.

[0004] The classification of proteases found in microorganisms is based on their catalytic mechanism, which is divided into four groups: serine proteases; metalloproteases; cysteine proteases; and aspartic proteases. These categories are:
They can be distinguished by their sensitivity to various inhibitors. For example, serine proteases are inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DIFP); metalloproteases are inhibited by chelators; cysteine enzymes are inhibited by iodoacetamide and heavy metals; aspartic proteases are inhibited by pepstatin Is done. Serine proteases have an alkaline pH optimum, metalloproteases are optimally active near neutrality, and cysteine and aspartic enzymes have an acidic pH optimum (Biotechnology Handbooks, Bacillus. Vol. 2, edited b
y Harwood, 1989 Plenum Press, New York).

[0005] The activity of cysteine proteases depends on cysteine and histidine catalytic pairs, which are different in order within the genus. The best-known family of cysteine proteases is that of papain, which has catalytic residues Cys-25 and His-159. The cysteine proteases of the papain family trigger the hydrolysis of peptide, amide, ester, thiol ester and thionoester bonds. Naturally occurring inhibitors of the papain family of cysteine proteases are cystatin family inhibitors (Methods in
Enxymology, vol.244, Academic Press, Inc. 1994).

[0006] onset Ming Overview invention, respectively, FIG. 1A-1B, having the nucleic acid and amino acid shown in FIG. 5A-5B and FIG. 6A-6B, referred to as CP1, CP2 and CP3 here, Bacillus It relates to the unexpected and surprising discovery of three previously unknown or unrecognized cysteine proteases found in bacteria. The present invention is based, in part, on the presence of the characteristic cysteine protease amino acid motif GXCWAF found in the uncharacterized translated genomic nucleic acid sequence of Bacillus subtilis. The invention is also based, in part, on the structural relevance of CP1 with the cysteine protease papain, particularly with respect to the location of catalytic histidine / alanine and asparagine / serine residues, and the structural relevance of CP1 with CP2 and CP3. Things.

[0007] The present invention provides isolated polynucleotide and amino acid sequences for CP1, CP2 and CP3. Due to the degeneracy of the genetic code, the invention encompasses nucleic acid sequences encoding the amino acid sequences of CP1, CP2 and CP3 shown in the figures.

The present invention provides a Bacillus subtilis CP1, CP disclosed herein having proteolytic activity.
2 and CP3 amino acid variants. Bacillus subtilis CP1, CP2 and CP3
, As well as proteolytically active amino acid variants, find use in cleaning compositions. In one embodiment of the present invention, CP1, CP obtained from Gram-positive microorganisms
2 or CP3 is produced on an industrial fermentation scale in a microbial host expression system. In another embodiment, an isolated and purified recombinant C obtained from a Gram-positive microorganism
P1, CP2 or CP3 is used in the composition of materials intended for cleaning purposes, such as cleaning agents. Accordingly, the present invention provides a cleaning composition comprising at least one of CP1, CP2 or CP3 obtained from a Gram-positive microorganism. The cysteine protease may be used alone in the cleaning composition or in combination with other enzymes and / or mediators or enhancers.

[0009] The production of the desired heterologous protein or polypeptide in Gram-positive microorganisms may be hindered by the presence of one or more proteases that degrade the produced heterologous protein or polypeptide. Thus, the present invention also relates to mutations of some or all of the genes encoding CP1 and / or CP2 and / or CP3 that will inactivate the proteolytic activity of CP1 and / or CP2 and / or CP3. Or deletion alone or, for example, apr,
Gram-positive microorganisms with other proteases, such as npr, epr, mpr, or with deletions or mutations in other proteases known to those of skill in the art are also included. In one embodiment of the present invention, the Gram-positive microorganism is a Bacillus bacterium. In another embodiment, the Bacillus is Bacillus subtilis.

[0010] In another embodiment, a Gram-positive microbial host having one or more deletions or mutations in a cysteine protease of the invention is further engineered to produce a desired protein. In one embodiment of the invention, the desired protein is atypical to said Gram-positive host cell. In another aspect, the desired protein is homologous to the host cell. The present invention relates to a gram having a nucleic acid encoding a homologous protein or a variant thereof that has been reintroduced in recombinant form with a deletion or interruption of a naturally occurring nucleic acid encoding a homologous protein such as a protease. Includes positive host cells. In another embodiment, the host cell produces a homologous protein. Accordingly, the present invention also provides a method and expression system for reducing the degradation of atypical or homologous proteins produced in Gram-positive microorganisms, wherein the nucleic acid encoding the atypical protein is a Bacillus host cell, Obtaining a host cell containing a mutation or deletion of at least one of the genes encoding cysteine protease 1, cysteine protease 2 and cysteine protease 3, and adapting the Bacillus host cell suitable for expression of the atypical protein Methods and expression systems comprising the steps of growing under conditions are provided. The Gram-positive microorganism may be sporulating or non-sporulating, as usual.

The present invention provides a method for detecting a Gram-positive microbial homolog of Bacillus subtilis CP1, CP2 and CP3, wherein a part or all of the nucleic acid encoding Bacillus subtilis CP1, CP2 and CP3 is replaced with a genome or cDNA. Hybridizing with nucleic acids from any source Gram-positive microorganism.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions As used herein, the genus Bacillus is not limited to,
B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alk
alophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautus and B
Includes all bacteria known to those skilled in the art, including .thuringiensis.

[0013] The present invention relates to new CP1, CP2 and CP3 from Gram-positive microorganisms. In a preferred embodiment, the Gram-positive microorganism is Bacillus. In another preferred embodiment, the Gram-positive microorganism is Bacillus subtilis. As used herein, "B. subtilis CP1, CP2 or CP3" refers to the amino acid sequence shown in the figure. 1A-1B show the amino acid and nucleic acid sequences of CP1 (YJDE), FIGS. 5A-5B show the amino acid and nucleic acid sequences of CP2 (YDHS), and FIGS. 6A-6B show the amino acid and nucleic acid sequences of CP3 (PMI). ing. The present invention includes amino acid variants of the amino acid sequences disclosed in FIGS. 1A-1B and FIGS. 5A-5B and FIGS. 6A-6B that have proteolytic activity. Such proteolytic amino acid variants can be used in cleaning compositions.

As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and its cross-section or portion, as well as the sensor or antisense strand.
DNA or RNA of genomic or synthetic origin, which may be double-stranded or single-stranded
. As used herein, "amino acid" refers to a peptide or protein sequence or portion thereof. A "polynucleotide homolog", as used herein, has at least 80%, at least 90% and at least 95% identity to Bacillus subtilis CP1, CP2 or CP3, or under conditions of high stringency. Refers to a Gram-positive microbial polynucleotide that can hybridize to Bacillus subtilis CP1, CP2 or CP3 and encodes an amino acid sequence having cysteine protease activity.

The term “isolated” or “purified” as used herein refers to a nucleic acid or amino acid that has been removed from at least one component with which it is naturally associated.

As used herein, “atypical protein” refers to a protein or polypeptide that is not naturally present in Gram-positive host cells. Examples of atypical proteins include hydrolases such as proteases, cellulases, amylases, carbohydrases, and lipases; racemases, epimerases, tautomers,
Or isomerases such as mutase; enzymes such as transferases, kinases and phosphatases. Variant genes may encode proteins or peptides that are important in therapy, such as growth factors, cytokines, ligands, receptors and inhibitors, and vaccines and antibodies. This gene contains protease, carbohydrase, amylase, glucoamylase, cellulase,
It may encode commercially important industrial proteins or peptides, such as oxidases and lipases. Genes of interest include naturally occurring genes,
It may be a mutated or synthetic gene.

“Homologous protein” refers to a protein or polypeptide that is innate or naturally occurring in Gram-positive host cells. The invention includes host cells that produce a homologous protein by recombinant DNA technology. The present invention relates to a nucleic acid encoding a reintroduced, homologous protein, or variant thereof, in recombinant form, with deletion or interruption of a naturally occurring nucleic acid encoding a homologous protein such as a protease. Gram-positive host cells having In another embodiment, the host cell produces a homologous protein.

As used herein, “overexpression” when referring to the production of a protein in a host cell means that the protein is produced in greater quantities than in a naturally occurring environment. .

As used herein, the term “proteolytic activity” refers to a protein that can hydrolyze peptide bonds. Enzymes having proteolytic activity are described in Enzyme Nomenclature, 1992, edited Webb Academic Press, Inc.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The unexpected discovery of the cysteine proteases CP1, CP2 and CP3 in Bacillus subtilis has led to the principle of producing host cells, to prevent the degradation of recombinantly produced heterologous proteins. Expression methods and systems that can be used are provided. In a preferred embodiment, the host cell has a deletion or inactivation in a naturally-occurring cysteine protease, wherein the mutation deletes or inactivates the production of the proteolytic cysteine protease gene product by the host cell. Gram-positive host cells. The host cell may be further genetically engineered to produce the desired protein or polypeptide.

It may be desirable to genetically manipulate any type of host cell to produce Gram-positive cysteine protease. Such host cells are isolated or purified and used in large-scale fermentations that produce large amounts of cysteine proteases used in cleaning products such as detergents.

I. Cysteine protease sequences CP1, CP2 and CP3 polynucleotides having the sequences shown in FIGS. 1A-1B, 5A-5B and 6A-6B, respectively, encode B. subtilis cysteine proteases CP1, CP2 and CP3. As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, various polynucleotides are
P2 and CP3 can be coded. The present invention includes all such polynucleotides.

The present invention provides that at least 80%, or at least 90%, or at least 95% identity to B. subtilis CP1, CP2 and CP3, so long as the homologue encodes a protein having proteolytic activity. CP1 encoding gram-positive microbial cysteine proteases CP1, CP2 and CP3, respectively
, CP2 and CP3 polynucleotide homologs.

A Gram-positive polynucleotide homolog of Bacillus subtilis CP1, CP2 or CP3 is
For example, from cloned DNA (eg, a DNA “library”), genomic DNA library, by chemical synthesis once identified, by cDNA cloning, or by the desired method, using standard methods known in the art. It may be obtained by cloning genomic DNA purified from cells or a fragment thereof (for example, see Sambrook et al., 1989, Molecular Cloning, A Laboratory Manua).
l, 2d Ed.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Y
ork; Glover, DM (ed.), 1985, DNA Cloning: A Practical Approach, MRL Pr
ess, Ltd. Oxford, UK Vol. I, II.). A preferred source is from genomic DNA. Nucleic acid sequences derived from genomic DNA may contain regulatory regions in addition to the coding region. Whatever its source, the isolated CP1, C
The P2 or CP3 gene should be molecularly cloned into a vector suitable for propagation of that gene.

In the molecular cloning of a gene from genomic DNA, DNA fragments are produced, some of which encode the desired gene. The DNA may be split at specific sites using various restriction enzymes. Alternatively, the DNA may be fragmented using DNase in the presence of manganese, or the DNA may be physically sheared, for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including, but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments have been produced, the identification of the specific DNA fragment containing CP1, CP2 or CP3 may be performed in various ways. For example, the Bacillus subtilis CP of the present invention
1. Isolate and label the CP2 or CP3 gene or its specific RNA, or a fragment thereof such as a probe or primer, and then use it in a hybridization assay to detect the Gram-positive CP1, CP2 or CP3 gene. (Benton, W. and Davis, R., 1977, Science 196: 180; Grunstein, M. And
Hogness, D., 1975, Proc. Natl. Acad. Sci. USA 72: 3961). Those DNA fragments that share substantial sequence similarity with the probe will hybridize under stringent conditions.

Accordingly, the present invention is a method for detecting Gram-positive CP1, CP2 and CP3 polynucleotide homologs, wherein a part or all of the nucleic acid sequence of Bacillus subtilis CP1, CP2 and CP3 is isolated from genomic or cDNA sources. The method comprising the step of hybridizing with the Gram-positive microorganism nucleic acid.

Also, under conditions of moderate to maximum stringency, Bacillus subtilis CP1, CP2
Alternatively, Gram-positive microbial polynucleotide sequences capable of hybridizing to the nucleotide sequence of CP3 are also included within the scope of the invention. Hybridization conditions are described in Berger and Kimmel (1987, Guide to Molecular Cloni, cited herein).
ng Techniques, Methods in Enzymology, Vol. 152, Academic Press, San Diego
As taught by CA), based on the melting temperature (Tm) of the nucleic acid binding complex, gives a "stringency" defined as described below.

“Maximum stringency” typically occurs at about Tm−5 ° C. (5 ° C. below the Tm of the probe); “High stringency” occurs at about 5 ° C. to 10 ° C. below Tm; "Medium stringency" occurs at about 10 to 20 ° C below Tm; "low stringency" occurs at about 20 to 25 ° C below Tm. As will be appreciated by those skilled in the art, maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences, while
Moderate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.

As used herein, “hybridization” includes “the process by which a nucleic acid strand binds to a complementary strand by base pairing” (Coombs J (1994) Dictionary of Biotechnolog).
y, Stockton Press, New York NY).

The process of amplification performed in the polymerase chain reaction (PCR) technique
effenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Col
d Spring Harbor Press, Plainview NY). A nucleic acid sequence of at least about 10 nucleotides to as many as about 60 nucleotides, preferably about 12 to 30 nucleotides, and more preferably about 20-25 nucleotides from Bacillus subtilis CP1, CP2 or CP3. Can be used as a probe or PCR primer.

The B. subtilis amino acid sequences CP1, CP2 and CP3 (shown in FIGS. 2, 4 and 3, respectively) were identified by a FASTA search of the B. subtilis genomic nucleic acid sequence. Bacillus subtilis CP1 (YJDE) was identified by its structural homology to the cysteine protease papain having a sequence designated "papa_carpa.p". As shown in FIG. 3, YJDE has the motif GXCWAF as well as the conserved catalytic residue His / Al
a and Asn / Ser. CP2 (YdHS) and CP3 (PMI)
Identified by structural homology to CP1 (YJDE). The presence of GXCWAF and the residues His / Ala and Asn / Ser are shown in FIGS. CP3 (P
MI) has been previously characterized as a potential phosphomannose isomerase (Noramata). CP3 has not been previously characterized as a cysteine protease.

II. Expression Systems The present invention provides host cells, expression methods and expression systems that provide improved production and secretion of desired heterologous or homologous proteins in Gram-positive microorganisms. In certain embodiments, the host cell has been genetically engineered to have a deletion or mutation in the gene encoding Gram-positive CP1, CP2 or CP3 such that the respective activity is deleted. In another embodiment of the present invention, the Gram positive microorganism has been genetically engineered to produce a cysteine protease of the present invention.

Inactivation of Gram-Positive Cysteine Protease in a Host Cell To produce an expression host cell that is unable to produce a naturally occurring cysteine protease, replacement of the naturally occurring gene from the genome of the host cell and / or
Or inactivation is required. In a preferred embodiment, the mutation is a non-reversion.

One method of mutating a nucleic acid encoding a Gram-positive cysteine protease involves cloning the nucleic acid or a portion thereof, mutating the nucleic acid by site-directed mutagenesis, and mutating the mutated nucleic acid. Is reintroduced into cells on a plasmid. The mutated gene may be introduced into the chromosome by homologous recombination. In the parent host cell, the result is that the naturally occurring nucleic acid and the mutated nucleic acid are tandemly located on the chromosome. After the second recombination, the mutated sequence remains in the chromosome, thereby effectively introducing the mutation into the chromosomal gene for the progeny of the parent host cell.

Another method of inactivating cysteine protease proteolytic activity is by deleting chromosomal gene copies. In a preferred embodiment, the entire gene is deleted and the deletion is made such that reversion is not possible. In another preferred embodiment, if the nucleic acid sequence left in the chromosome is too short for homologous recombination with a plasmid-encoded cysteine protease gene,
A partial deletion is made. In another preferred embodiment, the nucleic acid encoding the catalytic amino acid residue is deleted.

The deletion of a naturally occurring Gram-positive microbial cysteine protease may be performed as follows. The cysteine protease gene containing its 5 'and 3' terminal regions is isolated and inserted into a cloning vector. The coding region of the cysteine protease gene is deleted from the vector in vitro and a sufficient amount of 5 'to provide homologous recombination with the naturally occurring gene in the parent host cell.
And the 3 'terminal flanking sequence is left behind. This vector is then transformed into Gram-positive host cells. This vector is integrated into the chromosome by homologous recombination in the adjacent region. By this method, a Gram-positive strain in which the protease gene has been deleted is obtained.

[0038] The vector used in the integration method is preferably a plasmid. A selectable marker may be included to facilitate identification of the desired recombinant microorganism. Further, as will be appreciated by those skilled in the art, the vector is preferably one that can be selectively integrated into the chromosome. This can be done by introducing an inducible source of replication, eg, a temperature sensitive source, into the plasmid.
Growing the transformants at a temperature at which the origin of replication is sensitive inactivates the replication function of the plasmid, thereby providing a means for selecting chromosomal integrants. Integrants may be selected for growth at elevated temperatures in the presence of a selectable marker, such as an antibody substance. The embedding mechanism is described in International Patent Application Publication No.WO88 / 06623
No.

Integration by the Campbell-type mechanism occurs in the 5 ′ terminal flanking region of the protease gene, resulting in a protease-positive strain carrying the entire plasmid vector in the chromosome at the cysteine protease locus. Since different results can be obtained by illegitimate recombination, it is necessary to determine whether or not the complete gene has been deleted by nucleobase sequencing or a restriction map.

Another method of inactivating a naturally occurring cysteine protease gene is
The chromosomal inheritance of a gram-positive microorganism by transformation with a mutagenic oligonucleotide to mutagenize the copy. Or,
The chromosomal cysteine protease gene may be replaced with a mutant gene by homologous recombination.

The present invention relates to additional proteases such as deletions or mutations of the cysteine proteases of the invention and deletions or mutations in apr, pr, epr, mpr and others known to those skilled in the art. Includes host cells with deletions or mutations. U.S. Patent No. 5,264,366 discloses Bacillus host cells lacking apr and npr, and U.S. Patent No. 5,585,253 discloses Bacillus host cells lacking epr. , Margot et al., 1996, Microbiology 142: 3437-344
4 discloses a host cell lacking wpr, and EP 0369817 discloses a Bacillus host cell lacking mpr.

One assay for detecting mutants is to grow Bacillus host cells on media containing a protease substrate and measure the appearance or absence of rings around clear areas or colonies. Each step is included. Host cells with inactive proteases show little or no ring around the colony.

III. Cysteine Protease Production To produce cysteine protease in host cells, a Gram-positive microorganism C
An expression vector containing at least one copy, and preferably multiple copies, of a nucleic acid encoding P1, CP2 or CP3 is transformed into a host cell under conditions suitable for the expression of a cysteine protease. According to the present invention, a polynucleotide encoding a gram-positive microorganism CP1, CP2 or CP3 or a fragment thereof, or a fusion protein or polynucleotide homolog encoding an amino acid variant of Bacillus subtilis CP1, CP2 or CP3 is used. Thus, recombinant DNA molecules that support their expression in host cells may be produced. In a preferred embodiment, the Gram-positive host cell belongs to the genus Bacillus. In another preferred embodiment, said Gram-positive host cell is Bacillus subtilis.

As will be appreciated by those skilled in the art, it will be preferable to produce polynucleotide sequences that have non-naturally occurring codons. Codons preferred by particular Gram-positive host cells (Murray E et al. (1989) Nuc Acids Res 17: 477-508) are selected, for example, to increase expression rates or to be produced from naturally occurring sequences. A recombinant RNA transcript having desired properties, such as a longer half-life, can be produced than a transcript.

A modified CP1, CP2 or CP3 polynucleotide sequence that may be used in accordance with the present invention differs in that each results in a polynucleotide encoding the same or a functionally equivalent CP1, CP2 or CP3 homolog. Includes deletions, insertions or substitutions of nucleotide residues. As used herein, "deletion" is defined as a change in a nucleotide or amino acid sequence without one or more nucleotide or amino acid sequences, respectively.

As used herein, “insertion” or “addition” refers to naturally occurring CP1
, CP2 or CP3, respectively, means a change in the nucleotide or amino acid sequence at which one or more nucleotides or amino acid residues are added, respectively.

As used herein, “substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

The encoded protein undergoes a silent change and is functional CP1, CP
Deletions, insertions or substitutions of amino acid residues resulting in 2 or CP3 variants may be indicated. As long as the mutant retains the ability to modulate secretion, the polarity, charge,
Based on similarities in solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity,
Planned amino acid substitutions may be made. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine, as amino acids having an uncharged electrode head group with similar hydrophilicity. , Leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine, phenylalanine,
And tyrosine.

[0049] The CP1, CP2 or CP3 polynucleotides of the invention may be engineered to modulate the cloning, processing and / or expression of a gene product. For example, using techniques well known in the art, e.g., site-directed mutagenesis, mutations may be introduced, e.g., to insert new restriction sites, alter glycosylation patterns, Codon preferences may be changed.

In one embodiment of the invention, the gram-positive microorganism CP1, CP2 or C
A P3 polynucleotide may be ligated to a heterologous sequence to encode a fusion protein. The fusion protein may be engineered to include a split site located between the cysteine protease nucleotide sequence and the heterologous protein sequence to split the cysteine protease and purify from the heterologous portion.

VI. The expression vector used to express the cysteine protease of the present invention in a vector sequence Gram-positive microorganism comprises at least one promoter associated with a cysteine protease selected from the group consisting of CP1, CP2 and CP3, wherein the promoter is Functional in host cells. In one embodiment of the invention, the promoter is a wild-type promoter for a selected cysteine protease, and in another embodiment of the invention, the promoter is atypical to the cysteine protease. Is still functional in the host cell. In one preferred embodiment of the invention, the nucleic acid encoding the cysteine protease is stably integrated into the microbial genome.

[0052] In a preferred embodiment, the expression vector preferably includes a number of cloning site cassettes containing at least one restriction endonuclease site unique to the vector to facilitate nucleic acid manipulation. In a preferred embodiment, the vector also contains one or more selectable markers. As used herein, selectable marker refers to a gene that can be expressed in a Gram-positive host to facilitate selection of a host containing the vector. Examples of such selectable markers include, but are not limited to, antibiotics such as erythromycin, actinomycin, clamphenicol and tetracycline.

V. A variety of host cells can be used for the production of transformed CP1, CP2 and CP3, including bacterial, fungal, mammalian and insect cells. A common transformation method is Curren
t Protocols In Molecular Biology (vol.1, edited by Ausubel et al., John
Wiley & Sons, Inc. 1987, Chapter 9) and includes the calcium phosphate method, transformation with DEAE-dextran, and electroporation. Plant transformation methods are taught in Rodriquez (WO95 / 14099, published 26 May 1995).

[0054] In a preferred embodiment, the host cell is a Gram-positive microorganism, and in another preferred embodiment, the host cell is Bacillus. In one embodiment of the invention, a nucleic acid encoding one or more cysteine proteases of the invention is introduced into a host cell via an expression vector capable of replicating in a Bacillus host cell. A suitable replicating plasmid of the genus Bacillus has been
cular Biologial Methods for Bacillus, Ed.Harwood and Cutting, John Wile
y & Sons, 1990 (see chapter 3 for plasmids). Suitable replicating plasmids of Bacillus subtilis are listed on page 92.

In another embodiment, a nucleic acid encoding a cysteine protease of the invention is stably integrated into the genome of the microorganism. Preferred host cells are Gram-positive host cells. Another preferred host is Bacillus. Another preferred host is Bacillus subtilis. Several schemes have been described in the literature for direct cloning of DNA in Bacillus. Plasmid marker rescue transformation involves uptake of the donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al., Plasmid 2: 555-571 (1979); Haima
et al., Mol. Gen. Genet. 223: 185-191 (1990); Weinrauch et al., J. Bacterio
l. 154 (3): 1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169 (3): 12
05-1211 (1987)). The incoming donor plasmid recombines with the homologous region of the resident "helper" plasmid in a process similar to chromosomal transformation.

Transformation by protoplast transformation was described by Chang and Cohen, (197
9) Mol. Gen. Genet 168: 111-115; For B. metadata, see Vorobjeva et al., (
1980) FEMS Microbiol. Letters 7: 261-263; S for B. amyloliquefaciens
mith et al., (1986) Appl. and Env. Microbiol. 51: 634; for B. thuringiensis, Fisher et al., (1981) Arch. Microbiol. 139: 213-217; B. sphaericu
s is described in McDonald (1984) J. Gen. Microbiol. 130: 203; and B. larvae is described in Bakhiet et al., (1985) 49: 577. Mann et al., (1986, Current Microbiol. 13: 131-135) and Holubova (1985, Folia Microbiol. 30:97) on the transformation of Bacillus protoplasts include DNA containing liposomes. A method for introducing DNA into a protoplast using the method described above is disclosed.

VI. Identification of Transformants If the host cell has been transformed with a mutated or naturally occurring gene encoding Gram-positive CP1, CP2 or CP3, detection of the presence / absence of marker gene expression is of interest. It can be shown whether or not a certain gene is present. However, its expression should be confirmed. For example, if a nucleic acid encoding a cysteine protease has been inserted into the marker gene sequence, recombinant cells containing the insert can be identified by the absence of marker gene function. Alternatively, the marker gene may be arranged vertically under the control of one promoter and the nucleic acid encoding cysteine protease. Expression of the marker gene in response to induction or selection usually indicates expression of the cysteine protease as well.

Alternatively, host cells containing the coding sequence for a cysteine protease and expressing the protein may be identified by various methods known to those of skill in the art. These methods include, but are not limited to, DNA-DNA or DNA-RNA hybrids, including membrane-based, solution-based, or chip-based techniques for the detection and / or quantification of nucleic acids or proteins. Formation and protein bioassays or immunoassay techniques.

The presence of the cysteine polynucleotide sequence indicates that Bacillus subtilis CP1, CP2 or CP
DNA-DNA or DNA-RNA using 3 probes, parts or fragments
It can be detected by hybridization or amplification.

VII. Assays for Protease Activity Various assays for detecting and measuring protease activity are known to those skilled in the art.
There are assays based on the release of acid-soluble peptides from casein or hemoglobin, measured colorimetrically using the Folin method or as absorbance at 280 nm (
Bergmeyer et al., 1984, Methods of Enzymatic Analysis vol: 5, Peptidase,
Proteinases and their Inhibitors, Verlag Chemie, Weinheim). Other assays include solubilization of dye substrates (Ward, 1983, Proteinases, in Micr.
obial Enzymes and Biotechnology (WM Fogarty, ed.), Applied Science, Lo
ndon, pp. 251-317).

VIII. Measuring the level of secretion of the atypical or homologous protein in Gram-positive host cells;
Means for detecting a secreted protein include the use of either polyclonal or monoclonal antibodies specific for that protein. Such examples include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RI
A) and fluorescence-excited cell separation (FACS). These and other assays are described, inter alia, in Hampton R et al. (1990, Serological Methods, a Laboratory).
Manual, APS Press, St Paul MN) and Maddox DE et al. (1983, J Exp Med 1
58: 1211).

[0062] A variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for generating labeled hybridisation or PCR probes to detect a particular polynucleotide sequence include oligolabeling, nick translation, PCR amplification using end labels or labeled nucleotides. Alternatively, the nucleotide sequence, or some portion thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art and are commercially available;
7, may be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T3 or SP6 and a labeled nucleotide.

Many companies such as Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wyoming), and US Biochemical Corp (Cleveland, Ohio) Supplies commercial kits and experimental designs for these methods. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or dye agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Pat.
No. 837, No. 3,850,752, No. 3,939,350, No. 3,996,345, No. 4,277,437, No. 4,275,149 and No. 4,366,241. Recombinant immunoglobulins may also be produced as shown in U.S. Patent No. 4,816,567, cited herein.

IX. Protein Purification Gram-positive host cells transformed with a polynucleotide sequence encoding a variant or homologous protein may be cultured under conditions suitable for the expression and recovery of the encoded protein from the cell culture medium. The protein produced by the recombinant Gram-positive host cell containing the mutation or deletion of cysteine protease activity is secreted into the medium. Other recombinant constructs may link the atypical or homologous polynucleotide sequence to a nucleotide sequence encoding a polypeptide domain that facilitates the purification of soluble proteins (Kroll DJ et al. (1993) DNA Cell Bi
ol 12: 411-53).

Such purification-promoting domains include, but are not limited to, metal-chelating peptides such as a histidine-tryptophan module that can be purified on an immobilized metal (Porah J (1992) Protein Expr. Purif 3: 263-281), protein A domain that can be purified on immobilized immunoglobulin, and FL
Domains used in AGS extension / affinity purification system (Immunex: Immunex C
orp, Seattle, Washington). A splittable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the heterologous protein may be included to facilitate purification.

X. Uses of the Invention CP1, CP2 and CP3 and Genetically Engineered Host Cells The present invention provides for the use of CP1 such that its proteolytic activity is reduced or completely absent.
Genetically engineered host cells containing preferably non-reverting mutations or deletions in the naturally occurring gene encoding CP2 or CP3. The host cell may contain additional protease deletions, such as the deletion of a mature neutral protease and / or a mature subtilisin protease disclosed in US Pat. No. 5,264,366.

In a preferred embodiment, the host cell has been further genetically engineered to produce the desired protein or polypeptide. In a preferred embodiment, the host cell is of the genus Bacillus. In another preferred embodiment, the host cell is Bacillus subtilis.

[0068] In another embodiment, the host cell is a Gram-positive CP1, CP2 or CP
3 has been genetically engineered to produce 3. In a preferred embodiment, the host cells are grown under large-scale fermentation conditions and the CP1, CP2 or CP3
Is isolated and / or purified and used in a cleaning composition such as a detergent. Detergent formulations are disclosed in International Patent Application Publication No. WO 95/10615. The cysteine proteases of the present invention are useful for formulating various cleaning compositions. A variety of known compounds are suitable surfactants useful in compositions containing the cysteine proteases of the present invention. Examples of these include U.S. Pat.
Non-ionic, anionic, cationic, anionic or zwitterionic detergents as disclosed in U.S. Pat. Suitable cleaning compositions are described in U.S. Pat.
This is described in Example 7 of 4,015. The prior art is well aware of different formulations that can be used as cleaning compositions. Further, the use of the cysteine proteases of the invention in, for example, bar soap or liquid soap applications, dishwashing formulations, contact lens cleaning solutions or products, peptide hydrolysis, water treatment, textile applications, fusion split enzymes in protein products, etc. Can be. Cysteine proteases will provide improved performance in detergent compositions (as compared to other detergent proteases). As used herein, improved performance in detergents is due to increased detergency of certain enzyme-sensitive stains, such as grass or blood, as determined by normal evaluation after a standard wash cycle. Is defined as

The cysteine proteases of the present invention can be added to known powder or liquid detergents having a pH of from 6.5 to 12.0 at a level of from about 0.01% to about 5% by weight (preferably from 0.1% to 0.5%). Can be blended. These detergent compositions can include known proteases, amylases, cellulases, lipases or endoglycosidases, and builders and stabilizers.

The addition of cysteine protease to conventional cleaning compositions does not create any particular application limitations. In other words, any temperature and pH suitable for the detergent is suitable for the composition of the present invention, as long as the pH is in the range described above and the temperature is below the stated cysteine protease denaturation temperature. In addition, cysteine proteases, either alone or in combination with builders and stabilizers, can be used in detergent compositions without detergents.

One aspect of the present invention is a composition for treating textile comprising a cysteine protease of the present invention. This composition can also be used to treat, for example, silk or wool as described in publications such as RD216,034, EP 134,267, U.S. Pat.No. 4,533,359, and EP 344,259. No problem.

Proteases are disclosed in US Pat. Nos. 5,612,055, 5,314,692 and 5,147,
As described in 642, animal feed may be included as part of the animal feed additive.

The CP1, CP2 and CP3 polynucleotides of Bacillus subtilis polypeptide, or any portion thereof, provide the principle of detecting the presence of Gram-positive microbial polynucleotide homologs by hybridization and PCR techniques.

Thus, one aspect of the present invention provides nucleic acid hybridization and PCR probes that can be used to detect polynucleotide sequences, including genomic and cDNA encoding Gram-positive CP1, CP2 or CP3 or portions thereof. provide.

The manner and methods of practicing the present invention will be more fully understood by those skilled in the art with reference to the following examples. These examples are not intended to limit the scope of the invention or the claims in any way.

Example 1 Preparation of a Genomic Library The following example illustrates the preparation of a Bacillus library.

[0077] Genomic DNA from Bacillus cells was obtained by Current Protocols In Molocular Bio.
logy vol.1, edited by Ausubel et al., John Wiley & Sons, Inc. 1987, chap
ter Prepared as taught in 2.4.1. Generally, Bacillus cells from a saturated liquid medium are lysed and proteins are removed by digestion with proteinase K. Cell wall debris, polysaccharides, and residual protein are removed by selective precipitation with CTAB, and high molecular weight genomic DNA is recovered from the supernatant obtained by isopropanol precipitation. If extra clear genomic DNA is desired, an additional step of purifying the Bacillus genomic DNA with a cesium chloride gradient is added.

After obtaining purified genomic DNA, the DNA is subjected to Sar3A digestion. Sar3A is 4
It recognizes one base pair GATC and produces fragments compatible with several convenient phage lambda and cosmid vectors. The DNA is subjected to partial digestion to increase the chance of obtaining random fragments.

The partially digested Bacillus genomic DNA is subjected to size fractionation on a 1% agarose gel before cloning into a vector. Alternatively, size fractionation on a sucrose gradient can be used. The genomic DNA obtained from this size fractionation step is purified from agarose, ligated into a cloning vector suitable for use in a host cell, and transformed into the host cell.

Example II Detection of Gram-Positive Microorganisms The following example illustrates the detection of Gram-positive microorganisms CP1. CP2 and CP3 can be detected using the same method.

DNA derived from Gram-positive microorganisms was obtained by Current Protocols in Molecular Biology.
, Chap. 2 or 3. This nucleic acid was subjected to hybridization with a probe or primer derived from CP1 and / or PCR amplification. Preferred probes include a nucleic acid moiety that contains the conserved motif GXCWAF.

The nucleic acid probe was prepared by combining 50 picomoles of nucleic acid and 250 mCi of [gamma ³² P] adenosine triphosphate (Amersham, Chicago, Ill.) And T4 polynucleotide kinase (Dupont NEN®, Boston, Mass.)
) Are combined. The labeled probe is purified on a Sephadex G-25 ultrafine resin column (Pharmacia). Genome or c
For a typical membrane-based hybridization analysis of nucleic acid samples of any DNA origin,
Using a portion each containing per minute 10 ⁷ counts.

Restriction endonuclease digested DNA samples were fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plu, Durham, NH).
s, Schleicher & Schuell). Hybridization is carried out at 40 ° C. for 16 hours. The blot is subsequently washed at room temperature under increased stringency conditions to 0.1 × saline sodium citrate and 0.5% sodium dodecyl sulfate to remove non-specific signals. Exposing the blot to film for several hours,
The film is developed and the hybridization patterns are visually compared to detect polynucleotide homologs of Bacillus subtilis CP1. The nucleotide sequence of this homologue is determined for confirmation. Methods for nucleobase sequencing are well known in the art. In conventional enzymatic methods, DNA polymerase Klenow fragment SEQUENAS
DNA strands are extended from oligonucleotide primers annealed to the DNA template of interest using E® (US Biochemical, Inc., Cleveland, Ohio) or Taq polymerase.

Various other examples and modifications of the above description and examples will be apparent to those skilled in the art after reading this disclosure without departing from the spirit and scope of the present invention. Is intended to be included within the scope of the appended claims. All publications and patents referred to herein are fully incorporated herein by reference.

[Brief description of the drawings]

1A and 1B show the DNA (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of CP1 (YJDE).

FIG. 2 shows the YJDE of papain (SEQ ID NO: 3) (access number papa_carpa.p).
1 shows amino acid alignment with cysteine protease CP1. FIG.
For, 3 and 4, the motif GXCWAF is marked with catalytic cysteine and conserved catalytic histidine / alanine and asparagine / serine residues.

FIG. 3 shows the amino acid sequence alignment of CP1 (YJDE) (SEQ ID NO: 2) with CP3 (PMI) (SEQ ID NO: 5).

FIG. 4 shows the alignment of the amino acid sequence of CP1 (YJDE) (SEQ ID NO: 2) with CP2 (YdhS).

FIGS. 5A-5B show the amino acid (SEQ ID NO: 6) and nucleic acid sequence of CP2 (YdhS) (SEQ ID NO: 7).

FIGS. 6A-6B show amino acids (SEQ ID NO: 4) of CP3 (PMI) (SEQ ID NO: 5).
No.) and the nucleic acid sequence.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ, VN

Claims (17)

[Claims] 1. A Gram-positive microorganism in which a part or all of a gene encoding CP1 is mutated or deleted, and the mutation or deletion inactivates the proteolytic activity of the CP1. A gram-positive microorganism characterized by the fact that: 2. A Gram-positive microorganism in which a part or all of a gene encoding CP2 has been mutated or deleted, wherein the mutation or deletion inactivates the proteolytic activity of the CP2. A gram-positive microorganism characterized by the fact that: 3. A Gram-positive microorganism in which a part or all of a gene encoding CP3 is mutated or deleted, and the mutation or deletion inactivates the proteolytic activity of the CP3. A gram-positive microorganism characterized by the fact that: 4. The Gram-positive microorganism according to claim 1, which is a bacterium belonging to the genus Bacillus. 5. The method according to claim 5, wherein the bacterium is Bacillus subtilis, B. licheniformis, B. lentus, B. brevi.
s, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulan
The microorganism according to claim 4, wherein the microorganism is selected from the group consisting of s, B. ciculans, B. lautus, and B. thuringiensis. 6. The microorganism according to claim 1, wherein the microorganism is capable of expressing an atypical protein. 7. The microorganism according to claim 6, wherein said atypical protein is selected from the group consisting of hormones, enzymes, growth factors and cytokines. 8. The method according to claim 7, wherein the atypical protein is an enzyme.
The microorganism according to the above. 9. The method according to claim 8, wherein said enzyme is selected from the group consisting of proteases, carbohydrases and lipases; isomerases such as racemase, epimerase, tautomerase or mutase; transferases, kinases and phosphatases. The microorganism according to the above. 10. A cleaning composition comprising at least one cysteine protease selected from the group consisting of CP1, CP2 and CP3. 11. An expression vector comprising a nucleic acid encoding a cysteine protease selected from the group consisting of CP1, CP2 and CP3. 12. A host cell comprising the expression vector according to claim 11. 13. A method for producing an atypical protein in a Bacillus host cell, comprising: (a) comprising a nucleic acid encoding the atypical protein, encoding cysteine protease 1, cysteine protease 2 and cysteine protease 3; Obtaining a Bacillus host cell in which at least one of the following genes is mutated or deleted, and (b) growing the Bacillus host cell under conditions suitable for expression of the atypical protein. A method comprising: 14. The Bacillus host cell may be Bacillus subtilis, B. licheniformis, B.
.lentus, B.brevis, B.stearothermophilus, B.alkalophilus, B.amyloliquefac
14. The method according to claim 13, wherein the method is selected from the group consisting of iens, B. coagulans, B. ciculans, B. lautus and B. thuringiensis. 15. The Bacillus host cell may be apr, npr, epr, w
14. The method according to claim 13, wherein at least one of the genes encoding pr and mrp is mutated or deleted. 16. A Gram-positive microorganism in which at least one of the genes encoding cysteine protease selected from the group consisting of CP1, CP2 and CP3 is mutated or deleted. 17. The microorganism according to claim 16, wherein at least one of the genes encoding apr, npr, epr, wpr and mrp is mutated or deleted.