Note: Descriptions are shown in the official language in which they were submitted.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
1
PROTEASES FROM GRAM-POSITIVE ORGANISMS
FIELD OF THE INVENTION
s The present invention rela#es to cysteine proteases derived from gram-
positive microorganisms. The present invention provides nucleic acid and amino
acid
sequences of cysteine protease 1, 2 and 3 identified in Bacillus. The present
invention also
provides methods for the production of cysteine protease 1, 2 and 3 in host
cells as well as
the production of heterologous proteins in a host cell having a mutation or
deletion of part or
,o all of at least one of the cysteine proteases of the present invention.
BACKGROUND OF THE INVENTION
Gram-positive microorganisms, such as members of the group Bacillus, have been
used for large-scale industrial fermentation due, in part, to their ability to
secrete their
,s fermentation products into the culture media. In gram-positive bacteria,
secreted proteins
are exported across a cell membrane and a cell wall, and then are subsequently
released
into the external media usually maintaining their native conformation.
Various gram-positive microorganisms are known to secrete extracellular and/or
intracellular protease at some stage in their life cycles. Many proteases are
produced in
20 large quantities for industrial purposes. A negative aspect of the presence
of proteases in
gram-positive organisms is their contribution to the overall degradation of
secreted
heterologous or foreign proteins.
The classification of proteases found in microorganisms is based on their
catalytic
mechanism which results in four groups: the serine proteases;
metalloproteases; cysteine
zs proteases; and aspartic proteases. These categories can be distinguished by
their
sensitivity to various inhibitors. For example, the serine proteases are
inhibited by
phenylmethylsulfonylfluoride (PMSF} and diisopropylfluorophosphate (DIFP); the
metalloproteases by chelating agents; the cysteine enzymes by iodoacetamide
and heavy
metals and the aspartic proteases by pepstatin. The serine proteases have
alkaline pH
so optima, the metalloproteases are optimally active around neutrality, and
the cysteine and
aspartic enzymes have acidic pH optima (Biotechnology Handbooks, Bacillus.
vol. 2, edited
by Harwood, 1989 Plenum Press, New York).
The activity of cysteine protease depends on a catalytic dyad of cysteine and
histidine with the order differing among families. The best known family of
cysteine
ss proteases is that of papain having catalytic residues Cys-25 and His-159.
Cysteine
. proteases of the papain family catalyze the hydrolysis of peptide, amide,
ester, thiol ester
and thiono ester bonds. Naturally occurring inhibitors of cysteine proteases
of the papain
family are those of the cystatin family (Methods in Enzymology, vol. 244,
Academic Press,
Inc. 1994).
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
2
SUMMARY OF THE INVENTION
The present invention relates to the unexpected and surprising discovery of
three
heretofore unknown or unrecognized cysteine proteases found in Bacillus
subtilis,
designated herein as CP1, CP2 and CP3, having the nucleic acid and amino acid
as shown
in Figures 1A-1 B, Figures 5A-5B and 6A-6B, respectively. The present
invention is based, in
part, upon the presence of the characteristic cysteine protease amino acid
motif GXCWAF
found in uncharacterised translated genomic nucleic acid sequences of Bacillus
subtilis.
The present invention is also based in part upon the structural relatedness
that CP1 has
with the cysteine protease papain specifically with respect to the location of
the catalytic
.o histidine/alanine and asparagine/serine residues and the structural
relatedness that CP1
has with CP2 and CP3.
The present invention provides isolated polynucleotide and amino acid
sequences
for CP1, CP2 and CP3. Due to the degeneracy of the genetic code, the present
invention
encompasses any nucleic acid sequence that encodes the CP1, CP2 and CP3 amino
acid
,s sequence shown in the Figures.
The present invention encompasses amino acid variations of B.subtilis CP1, CP2
and CP3 amino acids disclosed herein that have proteolytic activity. B.
subtilis CP1, CP2
and CP3, as well as proteolytically active amino acid variations thereof, have
application in
cleaning compositions. In one aspect of the present invention, CP1, CP2 or CP3
obtainable
2o from a gram-positive microorganism is produced on an industrial
fermentation scale in a
microbial host expression system. In another aspect, isolated and purified
recombinant
CP1, CP2 or CP3 obtainable from a gram-positive microorganism is used in
compositions of
matter intended for cleaning purposes, such as detergents. Accordingly, the
present
invention provides a cleaning composition comprising at least one of CP1, CP2
and CP3
zs obtainable 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.
The production of desired heterologous proteins or polypeptides in gram-
positive
microorganisms may be hindered by the presence of one or more proteases which
degrade
so the produced heterologous protein or polypeptide. Therefore, the present
invention also
encompasses gram-positive microorganism having a mutation or deletion of part
or all of the
gene encoding CP1 and/or CP2 and/or CP3, which results in the inactivation of
the CP1
and/or CP2 and/or CP3 proteolytic activity, either alone or in combination
with deletions or
mutations in other proteases, such as apr, npr, epr, mpr for example, or other
proteases
ss known to those of skill in the art. In one embodiment of the present
invention, the gram-
positive organism is a member of the genus Bacillus. In another embodiment,
the Bacillus
is Bacillus subtilis.
In another aspect, the gram-positive microorganism host having one or more
deletions or mutations in a cysteine protease of the present invention is
further genetically
CA 02296708 2000-O1-11
WO 99/04016 PCT/IlS98/14529
3
engineered to produce a desired protein. In one embodiment of the present
invention, the
desired protein is heterologous to the gram-positive host cell. In another
embodiment, the
desired protein is homologous to the host cell. The present invention
encompasses a gram-
positive host cell having a deletion or interruption of the naturally
occurring nucleic acid
s encoding the homologous protein, such as a protease, and having nucleic acid
encoding
the homologous protein or a variant thereof re-introduced in a recombinant
form. In another
embodiment, the host cell produces the homologous protein. Accordingly, the
present
invention also provides methods and expression systems for reducing
degradation of
heterologous or homologous proteins produced in gram-positive microorganisms
comprising
,o the steps of obtaining a Bacillus host cell comprising nucleic acid
encoding said
heterologous protein wherein said host cell contains a mutation or deletion in
at least one of
the genes encoding cysteine protease 1, cysteine protease 2 and cysteine
protease 3; and
growing said Bacillus host cell under conditions suitable for the expression
of said
heterologous protein. The gram-positive microorganism may be normally
sporulating or
,s non-sporulating.
The present invention provides methods for detecting gram positive
microorganism
homologs of B. subtilis CP1, CP2 and CP3 that comprises hybridizing part or
all of the
nucleic acid encoding B. subtilis CP1, CP2 and CP3 with nucleic acid derived
from gram-
positive organisms, either of genomic or cDNA origin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1A-1B shows the DNA {SEA ID N0:1) and amino acid sequence for CP1
(YJDE) (SEQ ID N0:2).
Figure 2 shows an amino acid alignment with papain {SEQ ID N0:3) (accession
number papa_carpa.p) with the cysteine protease CP1, designated YJDE. For
Figures 2, 3
and 4, the motif GXCWAF has been marked along with the catalytic cysteine and
the
conserved catalytic histidinelalanine and asparagine/serine residues.
Figure 3 shows amino acid alignment of CP1 (YJDE) (SEQ ID N0:2) with CP3 (PMI)
(SEQ ID N0:5).
Figure 4 shows the amino acid alignment of CP1 (YJDE) (SEQ ID N0:2) with CP2
(YdhS).
Figure 5A-5B shows the amino acid (SEQ lD N0:6) and nucleic acid sequence for
CP2 {YdhS) (SEQ ID N0:7).
Figure 6A-6B shows the amino acid (SEQ ID N0:4) and nucleic acid sequence for
35 CP3 (PMI) (SEQ ID N0:5).
RECTIFIED SHEET (RULE 91)
ISA/EP
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/I4529
4
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
As used herein, the genus Bacillus includes all members known to those of
skill in
the art, including but not limited to B. subtilis, B. licheniformis, B.
lentos, B. brevis, B.
stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B.
ciculans, B.
lautus and B. thuringiensis.
The present invention relates to novel CP1, CP2 and CP3 from gram positive
organisms. In a preferred embodiment, the gram-positive organisms is a
Bacillus. In
another preferred embodiment, the gram-positive organism is Bacillus subtilis.
As used
,° herein, "B.subtilis CP1, CP2 or CP3" refers to the amino acid
sequences shown in Figures.
Figures 1A-1 B show the amino acid and nucleic acid seqeunce for CP1 (YJDE);
Figures 5A-
5B show the amino acid and nucleic acid sequence for CP2 (YDHS); and Figures
6A-6B
show the amino acid and nucleic acid sequences for CP3 (PMI). The present
invention
encompasses amino acid variations of the amino acid sequences disclosed in
Figures 1A-
,s 1 B and 5A-5B and 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
fragments or portions thereof, and to DNA or RNA of genomic or synthetic
origin which may
be double-stranded or single-stranded, whether representing the sense or
antisense strand.
zo As used herein "amino acid" refers to peptide or protein sequences or
portions thereof. A
"polynucleotide homolog" as used herein refers to a gram-positive
microorganism
polynucleotide that has at least 80%, at least 90% and at least 95% identity
to B.subtilis
CP1, CP2 or CP3, or which is capable of hybridizing to B.subtilis CP1, CP2 or
CP3 under
conditions of high stringency and which encodes an amino acid sequence having
cysteine
zs protease activity.
The terms "isolated" or "purified" as used herein refer to a nucleic acid or
amino acid
that is removed from at least one component with which it is naturally
associated.
As used herein, the term "heterologous protein" refers to a protein or
polypeptide
that does not naturally occur in a gram-positive host cell. Examples of
heterologous
3o proteins include enzymes such as hydrolases including proteases,
cellulases, amylases,
carbohydrases, and lipases; isomerases such as racemases, epimerases,
tautomerases, or
mutases; transferases, kinases and phophatases. The heterologous gene may
encode
therapeutically significant proteins or peptides, such as growth factors,
cytokines, ligands,
receptors and inhibitors, as well as vaccines and antibodies. The gene may
encode
35 commercially important industrial proteins or peptides, such as proteases,
carbohydrases
such as amylases and glucoamylases, cellulases, oxidases and lipases. The gene
of
interest may be a naturally occurring gene, a mutated gene or a synthetic
gene.
The term "homologous protein" refers to a protein or polypeptide native or
naturally
occurring in a gram-positive host cell. The invention includes host cells
producing the
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
homologous protein via recombinant DNA technology. The present invention
encompasses
a gram-positive host cell having a deletion or interruption of naturally
occurring nucleic acid
encoding the homologous protein, such as a protease, and having nucleic acid
encoding
the homologous protein, or a variant thereof, re-introduced in a recombinant
form. In
another embodiment, the host cell produces the homologous protein.
As used herein, the term "overexpressing" when refering to the production of a
protein in a host cell means that the protein is produced in greater amounts
than its
production in its naturally occurring environment.
As used herein, the phrase "proteolytic activity" refers to a protein that is
able to
,o hydrolyze a peptide bond. 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
B.subtilis
,s provides a basis for producing host cells, expression methods and systems
which can be
used to prevent the degradation of recombinantly produced heterologous
proteins. In a
preferred embodiment, the host cell is a gram-positive host cell that has a
deletion or
mutation in the naturally occurring cysteine protease said mutation resulting
in deletion or
inactivation of the production by the host cell of the proteofytic cysteine
protease gene
zo product. The host cell may additionally be genetically engineered to
produced a desired
protein or polypeptide.
It may also be desired to genetically engineer host cells of any type to
produce a
gram-positive cysteine protease. Such host cells are used in large scale
fermentation to
produce large quantities of the cysteine protease which may be isolated or
purified and
zs used in cleaning products, such as detergents.
I. Cysteine Protease Seguences
The CP1, CP2 and CP3 polynucleotides having the sequences as shown in Figures
1A-1B, 5A-5B and 6A-6B, respectively, encode the Bacillus subfilis cysteine
proteases CP1,
3o CP2 and CP3. As will be understood by the skilled artisan, due to the
degeneracy of the
genetic code, a variety of polynucleotides can encode the Bacillus subtilis
CP1, CP2 and
CP3. The present invention encompasses all such polynucleotides.
The present invention encompasses CP1, CP2 and CP3 polynucleotide homologs
encoding gram-positive microorganism cysteine proteases CP1, CP2 and CP3,
respectively,
ss which have at least 80%, or at least 90% or at least 95% identity to
B.subfilis CP1, CP2 and
CP3 as long as the homolog encodes a protein that has proteolytic activity.
Gram-positive polynucleotide homologs of B.subtilis CP1, CP2 or CP3 may be
obtained
by standard procedures known in the art from, for example, cloned DNA (e.g., a
DNA "library"),
genomic DNA libraries, by chemical synthesis once identified, by cDNA cloning,
or by the
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
6
cloning of genomic DNA, or fragments thereof, purified from a desired cell.
(See, for example,
Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.}, 1985, DNA
Cloning: A
Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) A preferred
source is from
genomic DNA. Nucleic acid sequences derived from genomic DNA may contain
regulatory
regions in addition to coding regions. Whatever the source, the isolated CP1,
CP2 or CP3
gene should be molecularly cloned into a suitable vector for propagation of
the gene.
In the molecular cloning of the gene from genomic DNA, DNA fragments are
generated,
some of which will encode the desired gene. The DNA may be cleaved at specific
sites using
,o various restriction enzymes. Alternatively, one may use DNAse in the
presence of manganese
to fragment the DNA, or the DNA can be physically sheared, as 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.
,s Once the DNA fragments are generated, identification of the specific DNA
fragment
containing the CP1, CP2 or CP3 may be accomplished in a number of ways. For
example,
a B.subtilis CP1, CP2 or CP3 gene of the present invention or its specific
RNA, or a
fragment thereof, such as a probe or primer, may be isolated and labeled and
then used in
hybridization assays to detect a gram-positive CP1, CP2 or CP3 gene. (Benton,
W. and
2o Davis, R., 1977, Science 196:180; Grunstein, M. And Hogness, D., 1975,
Proc. Natl. Acad.
Sci. USA 72:3961). Those DNA fragments sharing substantial sequence similarity
to the
probe will hybridize under stringent conditions.
Accordingly, the present invention provides a method for the detection of gram
positive CP1, CP2 and CP3 polynucleotide homoiogs which comprises hybridizing
part or all
is of a nucleic acid sequence of B. subtilis CP1, CP2 and CP3 with gram-
positive
microorganism nucleic acid of either genomic or cDNA origin.
Also included within the scope of the present invention are gram-positive
microorganism polynucleotide sequences that are capable of hybridizing to the
nucleotide
sequence of B.subfilis CP1, CP2 or CP3 under conditions of intermediate to
maximal
so stringency. Hybridization conditions are based on the melting temperature
(Tm} of the
nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to
Molecular
Cloning Techniques, Methods in Enzvmoloav, Vol 152, Academic Press, San Dieao
CA)
incorporated herein by reference, and confer a defined "stringency" as
explained below.
"Maximum stringency" typically occurs at about Tm-5°C (5°C below
the Tm of the
35 probe); "high stringency" at about 5°C to 10°C below Tm;
"intermediate stringency" at about
10°C to 20°C below Tm; and "low stringency" at about 20°C
to 25°C below Tm. As will be
understood by those of skill in the art, a maximum stringency hybridization
can be used to
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98114529
7
identify or detect identical polynucleotide sequences while an intermediate or
low stringency
hybridization can be used to identify or detect poiynucleotide sequence
homologs.
The term "hybridization" as used herein shall include "the process by which a
strand
of nucleic acid joins with a complementary strand through base pairing"
(Coombs J (1994)
s Dictionary of Biotechnology, Stockton Press, New York NY).
The process of amplification as carried out in polymerase chain reaction (PCR)
technologies is described in Dieffenbach CW and GS Dveksler (1995, PCR Primer,
a
Laboratory Manual, Cold Spring Harbor Press, Plainview NY). A nucleic acid
sequence of
at least about 10 nucleotides and as many as about 60 nucleotides from B.
subtilis CP1,
,o CP2 or CP3 preferably about 12 to 30 nucleotides, and more preferably about
20-25
nucleotides can be used as a probe or PCR primer.
The B.subtilis amino acid sequences CP1, CP2 and CP3 (shown in Figures 2, 4
and
3, respectively) were identified via a FASTA search of Bacillus subtilis
genomic nucleic acid
sequences. B. subtilis CP1 (YJDE) was identified by its structural homology to
the cysteine
,S protease papain having the sequence designated "papa_carpa.p". As shown in
Figure 2,
YJDE has the motif GXCWAF as well as the conserved catalytic residues His/Ala
and
Asn/Ser. CP2 (YdHS) and CP3 (PMI) were identified upon their structural
homology to CP1
(YJDE). The presence of GXCWAF as well as residues His/Ala and Asn/Ser is
noted in
Figures 3 and 4. CP3 (PMI) was previously characterized as a possible
phosphomannose
zo isomerase, (Noramata). There has been no previous characterization of CP3
as a cysteine
protease.
(I. Expression Systems
The present invention provides host cells, expression methods and systems for
the
zs enhanced production and secretion of desired heterologous or homologous
proteins in
gram-positive microorganisms. In one embodiment, a host cell is genetically
engineered to
have a deletion or mutation in the gene encoding a gram-positive CP1, CP2 or
CP3 such
that the respective activity is deleted. In another embodiment of the present
invention, a
gram-positive microorganism is genetically engineered to produce a cysteine
protease of
3o the present invention.
Inactivation of a Aram-positive cysteineprotease in a host cell
Producing an expression host cell incapable of producing the naturally
occurring
cysteine protease necessitates the replacement and/or inactivation of the
naturally occurring
35 gene from the genome of the host cell. In a preferred embodiment, the
mutation is a non-
reverting mutation.
One method for mutating nucleic acid encoding a gram-positive cysteine
protease is
to clone the nucleic acid or part thereof, modify the nucleic acid by site
directed
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
8
mutagenesis and reintroduce the mutated nucleic acid into the cell on a
plasmid. By
homologous recombination, the mutated gene may be introduced into the
chromosome. In
the parent host cell, the result is that the naturally occurring nucleic acid
and the mutated
nucleic acid are located in tandem on the chromosome. After a second
recombination, the
modified sequence is left in the chromosome having thereby effectively
introduced the
mutation into the chromosomal gene for progeny of the parent host cell.
Another method for inactivating the cysteine protease proteolytic activity is
through
deleting the chromosomal gene copy. In a preferred embodiment, the entire gene
is
deleted, the deletion occurring in such as way as to make reversion
impossible. In another
,o preferred embodiment, a partial deletion is produced, provided that the
nucleic acid
sequence left in the chromosome is too short for homologous recombination with
a plasmid
encoded cysteine protease gene. In another preferred embodiment, nucleic acid
encoding
the catalytic amino acid residues are deleted.
Deletion of the naturally occurring gram-positive microorganism cysteine
protease
,5 can be carried out as follows. A cysteine protease gene including its 5'
and 3' regions is
isolated and inserted into a cloning vector. The coding region of the cysteine
protease gene
is deleted form the vector in vifro, leaving behind a sufficient amount of the
5' and 3'
flanking sequences to provide for homologous recombination with the naturally
occurring
gene in the parent host cell. The vector is then transformed into the gram-
positive host cell.
zo The vector integrates into the chromosome via homologous recombination in
the flanking
regions. This method leads to a gram-positive strain in which the protease
gene has been
deleted.
The vector used in an integration method is preferably a plasmid. A selectable
marker may be included to allow for ease of identification of desired
recombinant
2s microorgansims. Additionally, as will be appreciated by one of skill in the
art, the vector is
preferably one which can be selectively integrated into the chromosome. This
can be
achieved by introducing an inducible origin of replication, for example, a
temperature
sensitive origin into the piasmid. By growing the transformants at a
temperature to which
the origin of replication is sensitive, the replication function of the
plasmid is inactivated,
3o thereby providing a means for selection of chromosomal integrants.
Integrants may be
selected for growth at high temperatures in the presence of the selectable
marker, such as
an antibiotic. Integration mechanisms are described in WO 88/06623.
Integration by the Campbell-type mechanism can take place in the 5' flanking
region
of the protease gene, resulting in a protease positive strain carrying the
entire plasmid
ss vector in the chromosome in the cysteine protease locus. Since illegitimate
recombination
will give different results it will be necessary to determine whether the
complete gene has
been deleted, such as through nucleic acid sequencing or restriction maps.
Another method of inactivating the naturally occurring cysteine protease gene
is to
mutagenize the chromosomal gene copy by transforming a gram-positive
microorganism
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98114529
9
with oligonucleotides which are mutagenic. Alternatively, the chromosomal
cysteine
protease gene can be replaced with a mutant gene by homologous recombination.
The present invention encompasses host cells having deletions or mutations of
a
cysteine protease of the present invention as well as additional protease
deletions or
s mutations, such as deletions or mutations in apr, npr, epr, mpr and others
known to those of
skill in the art. United States Patent 5,264,366 discloses Bacillus host cells
having a
deletion of apr and npr; United States Patent 5,585,253 discloses Bacillus
host cells having
a deletion of epr; Margot et al., 1996, Microbiology 142: 3437-3444 disclose
host cells
having a deletion in wpr and EP patent 0369817 discloses Bacillus host cells
having a
,o deletion of mpr.
One assay for the detection of mutants involves growing the Bacillus host cell
on
medium containing a protease substrate and measuring the appearance or lack
thereof, of
a zone of clearing or halo around the colonies. Host cells which have an
inactive protease
will exhibit little or no halo around the colonies.
t5
III. Production of Cysteine Protease
For production of cysteine protease in a host cell, an expression vector
comprising at
least one copy of nucleic acid encoding a gram-positive microorganism CP1, CP2
or CP3,
and preferably comprising multiple copies, is transformed into the host cell
under conditions
zo suitable for expression of the cysteine protease. In accordance with the
present invention,
polynucieotides which encode a gram-positive microorganism CP1, CP2 or CP3, or
fragments thereof, or fusion proteins or polynucleotide homolog sequences that
encode
amino acid variants of B.subtilis CP1, CP2 or CP3, may be used to generate
recombinant
DNA molecules that direct their expression in host cells. In a preferred
embodiment, the
is gram-positive host cell belongs to the genus Bacillus. In another preferred
embodiment, the
gram positive host cell is B. subtilis.
As will be understood by those of skill in the art, it may be advantageous to
produce
polynucleotide sequences possessing non-naturally occurring codons. Codons
preferred by
a particular gram-positive host cell (Murray E et al (1989) Nuc Acids Res
17:477-508) can
be selected, for example, to increase the rate of expression or to produce
recombinant RNA
transcripts having desirable properties, such as a longer half-life, than
transcripts produced
from naturally occurring sequence.
Altered CP1, CP2 or CP3 polynucieotide sequences which may be used in
accordance with the invention include deletions, insertions or substitutions
of different
35 nucleotide residues resulting in a polynucleotide that encodes the same or
a functionally
equivalent CP1, CP2 or CP3 homolog, respectively. As used herein a "deletion"
is defined
as a change in either nucleotide or amino acid sequence in which one or more
nucleotides
or amino acid residues, respectively, are absent.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
As used herein an "insertion" or "addition" is that change in a nucleotide or
amino
acid sequence which has resulted in the addition of one or more nucleotides or
amino acid
residues, respectively, as compared to the naturally occurring CP1, CP3 or
CP3.
As used herein "substitution" results from the replacement of one or more
5 nucleotides or amino acids by different nucleotides or amino acids,
respectively.
The encoded protein may also show deletions, insertions or substitutions of
amino
acid residues which produce a silent change and result in a functionally CP1,
CP2 or CP3
variant. Deliberate amino acid substitutions may be made on the basis of
similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of
,o the residues as long as the variant retains the ability to modulate
secretion. For example,
negatively charged amino acids include aspartic acid and glutamic acid;
positively charged
amino acids include lysine and arginine; and amino acids with uncharged polar
head groups
having similar hydrophilicity values include leucine, isoteucine, valine;
glycine, alanine;
asparagine, glutamine; serine, threonine, phenylalanine, and tyrosine.
,s The CP1, CP2 or CP3 polynucleotides of the present invention may be
engineered
in order to modify the cloning, processing and/or expression of the gene
product. For
example, mutations may be introduced using techniques which are welt known in
the art,
eg, site-directed mutagenesis to insert new restriction sites, to alter
glycosylation patterns or
to change codon preference, for example.
2o In one embodiment of the present invention, a gram-positive microorganism
CP1,
CP2 or CP3 poiynucleotide may be ligated to a heterologous sequence to encode
a fusion
protein. A fusion protein may also be engineered to contain a cleavage site
located
between the cysteine protease nucleotide sequence and the heterologous protein
sequence, so that the cysteine protease may be cleaved and purified away from
the
Zs heterologous moiety.
IV. Vector Seauences
Expression vectors used in expressing the cysteine proteases of the present
invention in gram-positive microorganisms comprise at least one promoter
associated with a
3o cysteine protease selected from the group consisting of CP1, CP2 and CP3,
which promoter
is functional in the host cell. In one embodiment of the present invention,
the promoter is
the wild-type promoter for the selected cysteine protease and in another
embodiment of the
present invention, the promoter is heterologous to the cysteine protease, but
still functional
in the host cell. In one preferred embodiment of the present invention,
nucleic acid
35 encoding the cysteine protease is stably integrated into the microorganism
genome.
In a preferred embodiment, the expression vector contains a multiple cloning
site
cassette which preferably comprises at least one restriction endonuclease site
unique to the
vector, to facilitate ease of nucleic acid manipulation. In a preferred
embodiment, the vector
also comprises one or more selectable markers. As used herein, the term
selectable
CA 02296708 2000-O1-11
WO 99/04016 PCTNS98/14529
11
marker refers to a gene capable of expression in the gram-positive host which
allows for
ease of selection of those hosts containing the vector. Examples of such
selectable
markers include but are not limited to antibiotics, such as, erythromycin,
actinomycin,
chloramphenicol and tetracycline.
V. Transformation
A variety of host cells can be used for the production of CP1,
CP2 and CP3 including bacterial, fungal, mammalian and insects cells. General
transformation procedures are taught in Current Protocols In Molecular Biology
(vol. 1,
,o edited by Ausubel et al., John Wiley ~ Sons, Inc. 1987, Chapter 9) and
include calcium
phosphate methods, transformation using DEAE-Dextran and electroporation.
Plant
transformation methods are taught in Rodriquez (WO 95/14099, published 26 May
1995).
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 present
,s invention, nucleic acid encoding one or more cysteine protease(s) of the
present invention is
introduced into a host cell via an expression vector capable of replicating
within the Bacillus
host cell. Suitable replicating plasmids for Bacillus are described in
Molecular Biological
Methods for Bacillus, Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby
expressly
incorporated by reference; see chapter 3 on plasmids. Suitable replicating
plasmids for B.
zo subtilis are listed on page 92.
In another embodiment, nucleic acid encoding a cysteine protease(s) of the
present
invention is stably integrated into the microorganism genome. Preferred host
cells are
gram-positive host cells. Another preferred host is Bacillus. Another
preferred host is
Bacillus subtilis. Several strategies have been described in the literature
for the direct
25 cloning of DNA in Bacillus. Plasmid marker rescue transformation involves
the uptake of a
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. Bacteriol. 154(3):1077-1087 (1983); and Weinrauch
et al., J.
Bacteriol. 169(3):1205-1211 (1987)). The incoming donor plasmid recombines
with the
so homologous region of the resident "helper' plasmid in a process that mimics
chromosomal
transformation.
Transformation by protoplast transformation is described for B. subfilis in
Chang and
Cohen, (1979) Mol. Gen. Genet 168:111-115; for B.megaterium in Vorobjeva et
al., (1980)
FEMS Microbiol. Letters 7:261-263; for B. amyloliquefaciens in Smith et al.,
(1986) Appl.
35 and Env. Microbiol. 51:634; for B.thuringiensis in Fisher et al., (1981 )
Arch. Microbiol.
139:213-217; for B.sphaericus in McDonald (1984) J. Gen. Microbiol. 130:203;
and B.larvae
in Bakhiet et al., (1985) 49:577. Mann et al., (1986, Current Microbiol.
13:131-135) report
on transformation of Bacillus protoplasts and Holubova, (1985) Folia
Microbiol. 30:97)
disclose methods for introducing DNA into protoplasts using DNA containing
liposomes.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
12
VI. Identification of Transformants
Whether a host cell has been transformed with a mutated or a naturally
occurring
gene encoding a gram-positive CP1, CP2 or CP3, detection of the
presence/absence of
marker gene expression can suggests whether the gene of interest is present
However, its
s expression should be confirmed. For example, if the nucleic acid encoding a
cysteine
protease is inserted within a marker gene sequence, recombinant cells
containing the insert
can be identified by the absence of marker gene function. Alternatively, a
marker gene can
be placed in tandem with nucleic acid encoding the cysteine protease under the
control of a
single promoter. Expression of the marker gene in response to induction or
selection
,o usually indicates expression of the cysteine protease as well.
Alternatively, host cells which contain the coding sequence for a cysteine
protease
and express the protein may be identified by a variety of procedures known to
those of skill
in the art. These procedures include, but are not limited to, DNA-DNA or DNA-
RNA
hybridization and protein bioassay or immunoassay techniques which include
membrane-
,S based, solution-based, or chip-based technologies for the detection and/or
quantification of
the nucleic acid or protein.
The presence of the cysteine polynucleotide sequence can be detected by DNA-
DNA or DNA-RNA hybridization or amplification using probes, portions or
fragments of
B.subtilis CP1, CP2 or CP3.
VII. Assay of Protease Activity
There are various assays known to those of skill in the art for detecting and
measuring protease activity. There are assays based upon the release of acid-
soluble
peptides from casein or hemoglobin measured as absorbance at 280 nm or
colorimetrically
2s using the Folin method (Bergmeyer, et al., 1984, Methods of Enzymatic
Analysis vol. 5,
Peptidases, Proteinases and their Inhibitors. Verlag Chemie, Weinheim). Other
assays
involve the solubilization of chromogenic substrates (Ward, 1983, Proteinases,
in Microbial
Enzymes and Biotechnology (W.M. Fogarty, ed.), Applied Science, London, pp.
251-317).
so VIII. Secretion of Recombinant Proteins
Means for determining the levels of secretion of a heterologous or homologous
protein in a gram-positive host cell and detecting secreted proteins include,
using either
polyclonal or monoclonal antibodies specific for the protein. Examples include
enzyme-
linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent
activated
35 cell sorting (FACS). These and other assays are described, among other
places, 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 158:1211 ).
A wide variety of labels and conjugation techniques are known by those skilled
in the
art and can be used in various nucleic and amino acid assays. Means for
producing labeled
4o hybridization or PCR probes for detecting specific polynucleotide sequences
include
CA 02296708 2000-O1-11
WO 99/04016 PC'TNS98/14529
13
oligolabeling, nick translation, end-labeling or PCR amplification using a
labeled nucleotide.
Alternatively, the nucleotide sequence, or any portion of it, may be cloned
into a vector for
the production of an mRNA probe. Such vectors are known in the art, are
commercially
available, and may be used to synthesize RNA probes in vitro by addition of an
appropriate
s RNA polymerise such as T7, T3 or SP6 and labeled nucleotides.
A number of companies such as Pharmacia Biotech (Piscataway NJ), Promega
(Madison WI), and US Biochemical Corp (Cleveland OH) supply commercial kits
and
protocols for these procedures. Suitable reporter molecules or labels include
those
radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents
as well as
,o substrates, cofactors, inhibitors, magnetic particles and the like. Patents
teaching the use
of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437;
4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced as
shown
in US Patent No. 4,816,567 and incorporated herein by reference.
,5 IX. Purification of Proteins
Gram positive host cells transformed with polynucleotide sequences encoding
heterologous or homologous protein may be cultured under conditions suitable
for the
expression and recovery of the encoded protein from cell culture. The protein
produced by
a recombinant gram-positive host cell comprising a mutation or deletion of the
cysteine
zo protease activity will be secreted into the culture media. Other
recombinant constructions
may join the heterologous or homologous polynucleotide sequences to nucleotide
sequence
encoding a polypeptide domain which will facilitate purification of soluble
proteins (Kroll DJ
et al (1993) DNA Cell Biol 12:441-53).
Such purification facilitating domains include, but are not limited to, metal
chelating
zs peptides such as histidine-tryptophan modules that allow purification on
immobilized metals
(Porath J (1992) Protein Expr Purif 3:263-281), protein A domains that allow
purification on
immobilized immunoglobulin, and the domain utilized in the FLAGS
extension/affinity
purification system (lmmunex Corp, Seattle WA). The inclusion of a cleavable
linker
sequence such as Factor XA or enterokinase (Invitrogen, San Diego CA) between
the
purification domain and the heteroiogous protein can be used to facilitate
purification.
X. Uses of The Present Invention
CP1. CP2 and CP3 and Genetically En4ineered Host Cells
The present invention provides genetically engineered host cells comprising
3s preferably non-revertible mutations or deletions in the naturally occurring
gene encoding
CP1, CP2 or CP3 such that the proteolytic activity is diminished or deleted
altogether. The
host cell may contain additional protease deletions, such as deletions of the
mature subtilisn
protease and/or mature neutral protease disclosed in United States Patent No.
5,264,366.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
14
In a preferred embodiment, the host cell is further genetically engineered to
produce
a desired protein or polypeptide. In a preferred embodiment the host cell is a
Bacillus. In
another preferred embodiment, the host cell is a Bacillus subtilis.
In an alternative embodiment, a host cell is genetically engineered to produce
a
gram-positive CP1, CP2 or CP3. In a preferred embodiment, the host cell is
grown under
large scale fermentation conditions, the CP1, CP2 or CP3 is isolated and/or
purified and
used in cleaning compositions such as detergents. Detergent formulations are
disclosed in
WO 95/10615. A cysteine protease of the present invention can be useful in
formulating
various cleaning compositions. A number of known compounds are suitable
surfactants
,° useful in compositions comprising the cysteine protease of the
invention. These include
nonionic, anionic, cationic, anionic or zwitterionic detergents, as disclosed
in US 4,404,128
and US 4,261,868. A suitable detergent formulation is that described in
Example 7 of US
Patent 5,204,015. The art is familiar with the different formulations which
can be used as
cleaning compositions. In addition, a cysteine protease of the present
invention can be
,5 used, for example, in bar or liquid soap applications, dishcare
formulations, contact tens
cleaning solutions or products, peptide hydrolysis, waste treatment, textile
applications, as
fusion-cleavage enzymes in protein production, etc. A cysteine protease may
provide
enhanced performance in a detergent composition (as compared to another
detergent
protease). As used herein, enhanced performance in a detergent is defined as
increasing
zo cleaning of certain enzyme sensitive stains such as grass or blood, as
determined by usual
evaluation after a standard wash cycle.
A cysteine protease of the present invention can be formulated into known
powdered and liquid detergents having pH between 6.5 and 12.0 at levels of
about .01 to
about 5% (preferably .1 % to .5%) by weight. These detergent cleaning
compositions can
zs also include other enzymes such as known proteases, amylases, cellulases,
lipases or
endoglycosidases, as well as builders and stabilizers.
The addition of a cysteine protease 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
~o range, and the temperature is below the described cysteine protease
denaturing
temperature. In addition, a cysteine protease can be used in a cleaning
composition without
detergents, again either alone or in combination with builders and
stabilizers.
One aspect of the invention is a composition for the treatment of a textile
that
includes a cysteine protease of the present invention. The composition can be
used to treat
35 for example silk or wool as described in publications such as RD 216,034;
EP 134,267; US
4,533,359; and EP 344.259.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
Proteases can be included in animal feed such as part of animal feed additives
as
described in, for example, US 5,612,055; US 5,314,692; and US 5,147,642.
CP1 CP2 and CP3 Polynucleotides
A B.subtlis polynucleotide, or any part thereof, provides the basis for
detecting the
presence of gram-positive microorganism polynucleotide homologs through
hybridization
techniques and PCR technology.
Accordingly, one aspect of the present invention is to provide for nucleic
acid
hybridization and PCR probes which can be used to detect polynucleotide
sequences,
,o including genomic and cDNA sequences, encoding gram-positive CP1, CP2 or
CP3 or
portions thereof.
The manner and method of carrying out the present invention may be more fully
understood by those of skill in the art by reference to the following
examples, which
examples are not intended in any manner to limit the scope of the present
invention or of
,s the claims directed thereto
Example I
Preparation of a Genomic library
The following example illustrates the preparation of a Bacillus genomic
library.
Genomic DNA from Bacillus cells is prepared as taught in Current Protocols In
2o Molecular Biology vol. 1, edited by Ausubel et al., John Wiley & Sons, Inc.
1987, chapter 2.
4.1. Generally, Bacillus cells from a saturated liquid culture are lysed and
the proteins
removed by digestion with proteinase K. Cell wall debris, polysaccharides, and
remaining
proteins are removed by selective precipitation with CTAB, and high molecular
weight
genomic DNA is recovered from the resulting supernatant by isopropanol
precipitation. If
2s exceptionally clean genomic DNA is desired. an additional step of purifying
the Bacillus
genomic DNA on a cesium chloride gradient is added.
After obtaining purified genomic DNA, the DNA is subjected to Sau3A digestion.
Sau3A recognizes the 4 base pair site GATC and generates fragments compatible
with
several convenient phage lambda and cosmid vectors. The DNA is subjected to
partial
so 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 prior to cloning into a vector. Alternatively, size
fractionation on a sucrose
gradient can be used. The genomic DNA obtained from the size fractionation
step is
purified away from the agarose and ligated into a cloning vector appropriate
for use in a
3s host cell and transformed into the host cell.
CA 02296708 2000-O1-11
WO 99/04016 PCT/US98/14529
16
Example ll
Detection of gram-postive microorganisms
The following example describes the detection of gram-positive microorganism
CP1.
The same procedures can be used to detect CP2 and CP3.
DNA derived from a gram-positive microorganism is prepared according to the
methods disclosed in Current Protocols in Molecular Biology, Chap. 2 or 3. The
nucleic acid
is subjected to hybridization and/or PCR amplification with a probe or primer
derived from
CP1. A preferred probe comprises the nucleic acid section containing the
conserved motif
GXCWAF.
,o The nucleic acid probe is labeled by combining 50 pmol of the nucleic acid
and 250
mCi of [gamma 32P] adenosine triphosphate (Amersham, Chicago IL) and T4
polynucleotide kinase (DuPont NEN~, Boston MA). The labeled probe is purified
with
Sephadex G-25 super fine resin column (Pharmacia). A portion containing 107
counts per
minute of each is used in a typical membrane based hybridization analysis of
nucleic acid
,s sample of either genomic or cDNA origin.
The DNA sample which has been subjected to restriction endonuclease digestion
is
fractionated on a 0.7 percent agarose gel and transferred to nylon membranes
(Nytran Plus,
Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at
40 degrees
C. To remove nonspecific signals, blots are sequentially washed at room
temperature
under increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium
dodecyf sulfate. The blots are exposed to film for several hours, the film
developed and
hybridization patterns are compared visually to detect polynucleotide homologs
of B.subtilis
CP1. The homologs are subjected to confirmatory nucleic acid sequencing.
Methods for
nucleic acid sequencing are well known in the art. Conventional enzymatic
methods employ
z5 DNA polymerise Klenow fragment, SEQUENASE~ (US Biochemical Corp, Cleveland,
OH)
or Taq polymerise to extend DNA chains from an oligonucleotide primer annealed
to the
DNA template of interest.
Various other examples and modifications of the foregoing description and
examples
will be apparent to a person skilled in the art after reading the disclosure
without departing
3o from the spirit and scope of the invention, and it is intended that all
such examples or
odifications be included within the scope of the appended claims. All
publications and
patents referenced herein are hereby incorporated by reference in their
entirety.
