Psychrophilic bacteria have been known for a long time, and their existence can be confirme extensively in low temperature circumstances. For example, psyhrophilic bacteria can be isolated from soils, fishery products, milk products as well as artificial low temperature circumstances. Enzymes, such as protases, obtained from psychrophilic bacteria are expected to be cold-active enzymes having an optimal activity at low temperature. For example, a protase (CP70) isolated from Flavobacterium balustinum has about 50% or more of its maximum activity at 20°C, but is completely inactive after about 10 minutes at 50°C (see e. g., World Patent Application US 97/01148, (Hasan; published July 31,1997)).

Such cold active enzymes may be included in detergent compositions for use in cold temperature wash water. However, such enzymes are produced in relatively low quantities by naturally-occurring (e. g., wild-type) psychrophilic bacteria.

Based on the foregoing, there is a need for an alternative source for producing relatively larger quantities of cold-active protase. To this end, there is a need for a polynucleotide sequence encoding a cold-active protase for incorporation into a recombinant host, for subsequent production of the cold- active protase.

SUMMARY The subject invention relates to an isolated polynucleotide comprising a nucleotide sequence substantially as shown in SEQ ID NO: 1; or substantially

similar to a nucleotide sequence contained in a plasmid having all of the identifying characteristics of Deposit No. FERM BP-6154.

The subject invention further relates to an expression system comprising the polynucleotide.

The subject invention further relates to a process for producing an isolated cold active protase polypeptide.

These and other features, aspects, and avantages of the present invention will become evident to those skilled in the art from a reading of the present disclosure.

DETAILED DESCRIPTION While the specification conclues with claims which particularly point out and distinctly claim the invention, it is believed the present invention will be bettes understood from the following description.

All cited references are incorporated herein by reference in their entireties.

Citation of any reference is not an admission regarding any determination as to its availability as prior art to the claimed invention.

All percentages are by weight of total composition unless specifically stated otherwise.

All ratios are weight ratios unless specifically stated otherwise.

Herein,"comprising"means that other steps and other ingredients which do not affect the end result can be added. This term encompasses the terms "consisting of'and"consisting essentially of'.

Herein,"isolated", in reference to the polypeptide of the present invention or polynucleotide encoding the polypeptide, means that the polypeptide or polynucleotide exists apart from the complex cellular milieu in which it naturally occurs, and the polypeptide is expressible from the polynucleotide in a cell that does not naturally express it when operably linked to the appropriate regulatory sequences. Specifically, when applied to polynucleotides (e. g. , DNA),"isolated" <BR> <BR> <BR> indicates the DNA is substantially isolated with respect to (ive. , exists substantially apart from) the complex cellular milieu in which it naturally occurs, or is simply present in a different nucleic acids context from that in which it occurs in nature (for example, when cloned or in the form of a restriction fragment). Thus,. the polynucleotide or polypeptide of the invention may be present in a wide variety of vectors, and/or in any of a wide variety of host cells (or other milieu, such as buffers, viruses or cellular extracts), and/or in any variety of compositions; yet still be isolated in the sense used herein in that such

vector, host cell or composition is not part of the naturai environment of the polynucleotide or polypeptide.

Herein,"substantially as shown"or"substantially similar", with respect to a polynucleotide, means the same or sufficiently similar in structure or nucleotide sequence to encode the desired polypeptide or gene product; or with respect to a polypeptide, the same or sufficiently similar in structure or amino acid sequence to serve its principal function. In other words, a particular subject sequence (amino acid or nucleotide sequence), for example altered by mutagenesis, varies from a reference sequence by one or more substitutions, deletions or additions, the net effect of which is to retain biological activity of the reference polypeptide. Alternatively, nucleotide sequences and analogs are "substantially similar"to the specific nucleotide sequence disclosed herein if the nucleotide sequences, as a result of degeneracy in the genetic code, encode an amino acid sequence substantially similar to the reference amino acid sequence.

In addition,"substantially similar"means a polypeptide that will react with antibodies generated against the polypeptide or peptides derived from the polypeptide of the invention.

The present invention relates to newly identifie polynucleotides, the use of such polynucleotides, as well as the production of polypeptides encoded by the polynucleotides.

One embodiment of the present invention is contained in INVa F' (pBSIISK (+)-CP70), an E. coli strain carrying a plasmid containing DNA encoding a CP70 polypeptide. This material has been deposited at the Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology (1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan) on October 23,1997. The deposited strain has been assigne Deposit No. FERM BP-6154.

The biological deposit referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for purposes of Patent Procedure. Consequently, the deposit is available as required by the patent laws in countries wherein counterparts of this patent application, or its progeny, are filed. However, Applicants'granting of such permission to the depository to distribute samples of the deposit does not constitute an express or implied license to practice the invention claimed in any patent issuing on the subject patent application or any other patent. The deposit is provided merely as a convenience to those skilled in the art, and is not an

admission that the deposited material is essential to the practice of the present invention. The nucleotide sequence of the polynucleotide contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are incorporated herein by reference in the event of any conflit with any description of sequences herein. It is noted that one of ordinary skill in the art reproducing Applicants'work from the written disclosure can discover any such sequencing conflits using routine skill.

A. CP70 Polypeptide The polynucleotide of the present invention encodes a polypeptide having an amino acid sequence substantially as shown in SEQ ID NO: 2; or substantially similar to the amino acid sequence encoded by a nucleotide sequence contained in a plasmid having all the identifying characteristics of Deposit No. FERM BP- 6154 (hereinafter collectively referred to as"CP70 polypeptide").

Herein"polypeptide"refers to a polymer made up of amino acids linked together to form peptide bonds, preferably forming a preproprotein, proprotein, protein or fragment thereof. Herein"preproprotein"refers to a polypeptide consisting of a signal sequence, a pro region, and a mature region; and Uproprotein"refers to a polypeptide consisting of a pro region and a mature region. Depending upon the host employed in a recombinant production procedure, the CP70 polypeptide may be glycosylated or may be non- glycosylated. The CP70 polypeptide may also include an initial methionine amino acid residue.

The CP70 polypeptide has an optimal temperature in a low temperature range. Consequently, the CP70 polypeptide permits the decomposition rection of a protein to be carried out in low temperature environments. For example, a detergent utilizable even in low temperature water can be prepared by adding the CP70 polypeptide to the detergent composition. Such a detergent composition can be prepared in accordance with methods and formulations well known in the art, except that the CP70 polypeptide produced by the polynucleotide of the present invention is added. In other words, the detergent composition can be formed by blending the CP70 polypeptide with ordinary detergent components such as surfactant, bleach, builder and the like.

B. Polynucleotide One aspect of the present invention relates to an isolated polynucleotide comprising a nucleotide sequence substantially as shown in SEQ ID NO: 1, substantially similar to a nucleotide sequence contained in a plasmid having all

of the identifying characteristics of Deposit No FERM BP-6154, or which encodes a CP70 polypeptide (hereinafter collectively referred to as"CP70 polynucleotide").

Herein,"polynucleotide"refers to a polymer of DNA or RNA which can be single-or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The polynucleotide may be in the form of a separate fragment or as a component of a larger nucleotide sequence construct, which has been derived from a nucleotide sequence isolated at least once in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated nucleotides may be present 5'or 3'from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions. Polynucleotides encoding the polypeptide provided by this invention can be assemble from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.

The polynucleotide of the present invention embraces nucleotide sequences having any sequence so long as it encodes the CP70 polypeptide.

As a result of degeneracy in the genetic code, any particular amino acid sequence may be encoded by many different nucleotide sequences. The skilled artisan will appreciate that the degeneracy of the genetic code allows for differing nucleotide sequences to provide the same polypeptide. In certain cases preparing a nucleotide sequence, which encodes for the same peptide but differs from the native nucleotide sequence, provides various advantages, including: ease of sequencing or synthesis, increased expression of the peptide, and/or preference of certain heterologous hosts for certain codons over others. These practical considerations are widely known and provide embodiments that may be advantageous to the user of the invention. Thus it is clearly contemplated that the native nucleotide sequence, or nucleotide sequence listed in the Sequence Listing, or incorporated by reference are not the only embodiments or nucleotide sequences envisioned by this invention.

Various embodiments of the CP70 polynucleotide inclue, but are not limited to, a nucleotide sequence as shown in: SEQ ID NO: 1; as well as positions 1-3413 of SEQ ID NO: 1 and positions 1-2533 of SEQ ID NO: 1.

Additional embodiments of the CP70 polynucleotide comprise nucleotide sequences which encode the"catalytic triad"of SEQ ID NO: 2, i. e. encode positions 30-39,66-75, and 315-332 of the polypeptide of SEQ ID: NO: 2. Stated another way, such embodiments comprise 764-793,872-901 and 1619-1672 of SEQ ID NO: 1; more preferably such an embodiment comprises positions 764- 1672 of SEQ ID NO: 1.

In another embodiment of the present invention, the CP70 polynucleotide is a nucleotide sequence which is at least about 60% homologous to the nucleotide sequence as shown in SEQ ID NO: 1, as shown in the above subsequences of SEQ ID NO: 1, or as contained in a plasmid having all of the identifying characteristics of Deposit No FERM BP-6154; more preferably at least about 75%; more preferably at least about 90%, more preferably at least about 95%; more preferably at least about 99%. Preferably such homologous sequences are capable of encoding a CP70 polypeptide.

C. Expression System Another aspect of the present invention relates to an expression system comprising the CP70 polynucleotide. Such expression systems include recombinant expression vectors comprising the CP70 polynucleotide, as well as hosts which have been genetically engineered with such recombinant expression vectors ("recombinant host").

1. Vector Herein,"recombinant expression vector"refers to a DNA construct used to express a polynucleotide which encodes a desired polypeptide (for example, the CP70 polypeptide) and which inclues a transcriptional subunit comprising an assembly of 1) genetic elements having a regulatory role in gene expression, for example, promoters and enhancers, 2) a structural or coding sequence which is transcribed into MARNA and translate into protein, and 3) appropriate transcription and translation initiation and termination sequences. Using methodology well known in the art, recombinant expression vectors of the present invention can be constructed. The nature of the vector is not critical to the invention, and any vector may be used, including plasmid, virus, bacteriophage, and transposon. Possible vectors for use in the present invention inclue, but are not limited to, chromosomal, nonchromosomal and synthetic DNA sequences, e. g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccina, adenovirus, fowl pox virus, and

pseudorabies. Additional useful vectors inclue, but are not limited to: for mammalian cells, pcDNA-1 (Invitrogen, San Diego, CA) and pSV-SPORT 1 (Gibco-BRL, Gaithersburg, MD); for insect cells, pBlueBac lil or pBlueBacHis baculovirus vectors (Invitrogen, San Diego, CA); and for bacterial cells, pET-3 (Novagen, Maison, WI). Any other vector may be used as well, as long as it is repliable and viable in the host. The CP70 polynucleotide can be present in the vector operably linked to regulatory elements.

The CP70 polynucleotide may be inserted into the vector by a variety of procedures. In general, the polynucleotide is inserted into an appropriate restriction endonuclease site (s) by procedures known in the art. Such procedures and others are deemed within the scope of those skilled in the art.

The vector may preferably comprise an expression element or elements operably linked to the CP70 polynucleotide to provide for expression thereof at suitable levels. Any of a wide variety of expression elements may be used. The expression element or elements may, for example, be selected from promoters, enhancers, ribosome binding sites, operators and activating sequences. Such expression elements may be regulatable, for example, inducible (via the addition of an inducer). Representative examples of useful promoters inclue, but are not limited to: LTR (long terminal repeat from a retrovirus) or SV40 promoteur, the E. coli lac or trp promoter, the phage Lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector preferably also contains a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression.

In a preferred embodiment, the expression vector further contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance for prokaryotic cell cultures.

Useful expression vectors for bacterial use are constructed by inserting a CP70 polynucleotide with suitable translation initiation and termination signals in operable reading frame with a functional promoter. The vector will preferably contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors inclue, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Maison, Wisconsin, U. S. A.). These pBR322 "backbone"sections are combine with an appropriate promoter and the CP70 polypeptide to be expressed. Other suitable bacterial vectors include: pQE70, pQE60, and pQE-9 (Qiagen); pbs, pD10, phagescript, psiX174, pBluescript SK, pbsks, pNH8A, pNH16a, pNH18A, and pNH47A (Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia).

Useful expression vectors for use with yeast can comprise a yeast replication origin or fragments of DNA which are required for integration into the host's chromosomal DNA, a selectable-marker, a suitable promoter and enhancer, and also any necessary ribosome binding sites, a polyadenilation site, transcriptional termination sequences and 5'flanking nontranscribed sequences.

Suitable yeast expression vectors inclue, but are not limited to, pPIC3, pPIC3K, pPIC3.5K, pPIC9, pPIC9K, pA0815, pHIL-D2, pHIL-S1, pPICZaA, pPICZaB, and <BR> <BR> <BR> pPICZaC (Invitrogen) preferably for Pichia pastoris; pYES2 (Invitrogen), and the pRS series vectors (STRATAGENE) preferably for Saccharomyces cerevisiae.

Mammalian expression vectors will preferably comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5'flanking nontranscribed sequences.

DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the necessary nontranscribed genetic elements.

Suitable mammalian vectors, by way of non-limiting example, include: pWLNEO, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia).

2. Host The recombinant expression vector containing the CP70 polynucleotide as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the recombinant host to express the CP70 polypeptide.

Recombinant hosts may include bacterial, fungai, insect, plant or mammalian cells which have been transformed with a recombinant expression

vector of the present invention. Recombinant hosts may also include entire plants, insects or non-human mammals which have been transformed with the recombinant expression vector. Representative examples of appropriate hosts for in vitro production include: bacterial cells such as E. coli, Salmonella typhimurium, Bacillus subtils, and various species within the general Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice; yeast or fungal cells such as Pichia pastoris, Candida boidinii, and Saccharamyces cervisiae; insect cells such as Drosophila and Sf9; animal cells such as the COS-7 lines of monkey kidney fibroblasts (described by Gluzman, CELL, 23: 175 (1981)), C127 (mouse), 3T3 (mouse), CHO (hamster) and BHK (hamster); human cells such as HeLa. Alternatively, recombinant hosts for in vivo production in non-human mammals inclue, but are not limited to, cows, goats, guinea pigs, hamsters, mice, pigs, rabbits and sheep; insects including silk worm larvae; and plants. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

Herein,"transformation"means introducing DNA into a cell or an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described by Cohen, S. N., PROC. NATL. ACAD.

Sci. (USA))) 69: 2110 (1972); Mandel et a/., J. MoL. BioL., 53: 154 (1970); and Lilgestrom et a/., GENE, 40: 241-246 (1985), is generally used for prokaryotes or other cells that contain substantial cell-wall barries. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, VIROLOGY, 52: 456-457 (1978) is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U. S. Pat. No. 4,399,216 issued Aug. 16,1983. Transformations into yeast are typically carried out according to the method of Van Solingen, ef al., J. BACT., 130: 946 (1977) and Hsiao, et a/. , PROC. NATL. ACAD. Sci. (USA), 76: 3829 (1979).

Alternatively, introduction of the expression vector into the host can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation as set forth in BASIC METHODS IN MOLECULAR BIOLOGY (D. L. Davis and I. M. Battey, (1986)). However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used.

D. Process for Producinq CP70 Polypeptide

One aspect of the invention relates to a process for producing an isolated CP70 polypeptide. The process comprises inserting the CP70 polynucleotide into a suitable recombinant expression vector (as described above), and then transforming a suitable host with this recombinant expression vector (as described above). The transformed host is subsequently used to express the CP70 polypeptide, followed by purification of the resulting CP70 polypeptide from the host.

In a preferred embodiment, the method comprises culturing a yeast host cell which has been transformed with a recombinant expression vector comprising the CP70 polynucleotide. The cultured bacterial host cell is subsequently used to express the CP70 polypeptide, followed by purification of the resulting CP70 polypeptide from the cultured medium.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e. g., temperature shift or chemical induction) and cells are cultured for an additional period. The cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of the CP70 polypeptide can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents; such methods are well known to those skilled in the art.

The CP70 polypeptide can be recovered and purifie from the recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The following examples further describe and demonstrate the preferred embodiments within the scope of the present invention. The examples are given solely for the purpose of illustration, and are not to be construed as limitations of the present invention since many variations thereof are possible without departing from its spirit and scope.

Example 1 This example shows the isolation of a CP70 polynucleotide. A genomic library of Flavobacterium balustinum P104 is constructed from partially digeste chromosomal DNA (with Sau3A) by ligating into the BamHl site of a lambda FIX 11 cloning vector. For the isolation of lambda clones carrying the CP70 gene, a pair of oligonucleotide primers are synthesized according to the N-terminal amino acid sequence of SEQ ID NO: 2. They are used to amplify a 90 base pair fragment corresponding to the amino acid sequence. This fragment was radioactively labeled with 32P and then used as a probe to screen lambda clones containing the CP70 gene. In one experiment, six positive clones were obtained <BR> <BR> from 8xi 03 phage plaques of the library. Further restriction mapping analysis indicated that the CP70 gene was located on a 3.4 kb EcoRl fragment within the insert DNA. Therefore, the 3.4 kb EcoRl fragment was isolated and subcloned into the pbluescript 11 SK+ vector for subsequent analysis. DNA sequencing analysis was performed with LI-COR model 4000L automatic DNA sequencer.

The CP70 gene was revealed to have the nucleotide sequence set forth in Deposit No FERM BP-6154 and in SEQ ID NO: 1.

Examnle 2 This example shows the chemical synthesis of a CP70 polynucleotide. A single-or double-stranded oligonucleotide which corresponds to any desired part of the CP70 gene of Example 1 is chemically synthesized with a nucleotide synthesizer such as ABI DNA/RNA synthesizer model 380A/380B/381 A/390Z/391/392/394.

Example 3 This example shows the formation of an expression vector containing a CP70 polynucleotide. Recombinant CP70 is designed to be expressed in the cultured media either as the mature form or with an artificial pro sequence. For the construction of the former expression vector, a combination of appropriate restriction enzyme sites (Xho1 and Not1) are introduced to the 5'and 3'ends of the mature portion of the CP70 polynucleotide by using the recombinant PCR technique. For the construction of the latter expression vector, the mature portion of the CP70 polynucleotide is fused to an artificial pro sequence containing a repeat of six histidine residues at the 5'end in addition to the restriction enzyme sites. This histidine tag is designed for a quick purification of the CP70 protein after the expression. The pro sequence also contains a specific cleavage site by

Enterokinase so that the recombinant pro CP70 can be activated by the Enterokinase digestion after the purification step.

These CP70 nucleotide cassettes are cloned between the Xho1 and Not1 sites of the pPIC9 vector (Invitrogen) for the expression in the yeast Pichia pastoris cells. The cloning sites are located immediately downstream of a yeast a-factor signal sequence in frame so that the recombinant mature-or pro-CP70 will be secreted into the medium. The induction of the CP70 protein is under control of the AOX1 promoter which lies upstream of the yeast a-factor signal sequence. The transcription from the AOX1 promoter is strongly induced by adding methanol in the medium and thus the recombinant CP70 protein can be induced. The vector also contains an Ampicillin resistance gene and a HIS4 gene for the selection in E. coli and Pichia pastoris cells, respectively.

Example 4 This example shows the transformation of a host with the expression system of Example 3. Transformation of Pichia cells is done by either the spheroplasts method or the electroporation method. (Refer to the manual of methods for expression of recombinant proteins in Pichia pastoris (catalog no.

K1710-01), which is distributed by Invitrogen). Pichia spheroplasts are prepared by treating Gus115, KM71 or SMD1168 strains with sorbitol and Zymolyase. 10 pg of the expression plasmid DNA is added to transform the spheroplast preparation in the presence of PEG and CaC12-Spheroplasts are then plated onto Histidine-depleted plates for the selection of HIS+ transformants at 28-30°C.

For the electroporation method, Pichia GS115, KM71 or SMD1168 cells are grown in rich media, collecte and treated with 1 M sorbitol solution. After mixing cells with 5-20 pg of linearized plasmid DNA, the solution is transferred to an ice-cold 0.2 cm electroporation cuvette (Bio-Rad). The electroporation is carried out at 1,500 volts with 25 pF capacitance and 200 Q resistance with a Bio-Rad Gene Pulsar. The cells are spread onto plates to obtain HIS+ transformants at 28-30 °C. Linear DNA can generate stable transformants of Pichia pastoris via homologous recombination between the transforming DNA and regions of homology within the genome. Single crossover events (insertion) are much more likely than double crossover events (replacements). Since single crossover events do not destroy the AOX1 gene on the host genome, such transformants can grow well on methanol-containing plates (HIS+ and Mut+ phenotype). In contrast, when the gene replacement arises from a double crossover event between the AOX1 promoter and 3'AOX1 regions of the vector

and gnome, this results in the complete removal of the AOX1 coding region.

The transformants with the resulting phenotype (HIS+ Mutes) can be selected by their slow growth on the methanol-containing plate. Both the Mut+ and Mutes transformants are screened for CP70 expression.

Example 5 This example shows the expression and purification of a CP70 polypeptide.

Expression: The transformants of Example 4 are grown in either minimal or rich media until they reach their maximum growth by either shake flask or fermentation. For the induction of the CP70 polypeptide, methanol is added in the medium at the maximum concentration of 1% in the case of the Muts transformants. With the Mut+ transformants, methanol is added at the levels which allow the cells to maintain the dissolve oxygen levels in the medium above 20%. About 6 days after starting the methanol induction, the medium is harvested and the supernatant is collecte by centrifugation.

Purification: The supernatant is applied onto a nickel-chelating column and the pro-CP70 is adsorbe through the high afffinity of the HIS tag for divalent cations. After washing unrelated proteins in the medium off the column, the pro- CP70 protein is recovered by elution in a low pH buffer or by competition with imidazole or histidine. The eluate is concentrated by ultrafiltration and the buffer is exchanged with the Enterokinase rection buffer. Activation of the pro-CP70 protein is carried out by treating it with Enterokinase. Enterokinase is removed by appropriate biochemical means after the activation step, and the sample is concentrated, lyophilized and kept at 4 OC until usage.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The Procter & Gamble Company, N/A N/A (ii) TITLE OF INVENTION: A Polynucleotide Encoding CP70 Cold Active Protase (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: T. David Reed (B) STREET: 5299 Spring Grove Avenue (C) CITY: Cincinnati (D) STATE: Ohio (E) COUNTRY: USA (F) ZIP: 45217-1087 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Reed, T David (B) REGISTRATION NUMBER: 32,931 (C) REFERENCE/DOCKET NUMBER: AA- (ix) TELECOMMUNI QTION INFORMATION: (A) TELEPHONE: 513-627-7025 (B) TELEFAX: 513-627-6333 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3413 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 437.. 2536 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 677.. 2536

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAATTCTTGC GGCGTTAAAT GGTATATCAT ACCCTTCCGG TTTTATTATA TAATCCTGAT 60 TTTCAAGCAA ATTACCATTA TCATCATGCA ATTGCCACGC AATCTGTACC ATTCTTGGCC 120 AGTTGTCTGA ATCTGATAAT GGAGCATTGA AATTTTTCGG TAAACCTGTG GTTTCTGTGT 180 CAAAAACTAA ATACATGTAA TTTTTTCCAG CTTTTATTAA AAAAAGAGAA TGTAAAGTTA 240 CTTCAATTTT TATAATTTAT TTCGATAACT TTATAAAAAC AACTTCTTTT TTAACATCAC 300 AAGAAGTACA TAAAAAATAA QATTTTAAC AAAAGAACQ AATATTTATT TACTAATTTA 360 ATAATTTTAC TCAAAGAAAG TTATTAATTC ATATATTTGC TTCTCATAAT CGTGTAATAA 420 TTTTTAATAA TCTATC ATG AAA AAA CAT TTA CTT TTA GTA AGT ACA TTG 469 Met Lys Lys His Leu Leu Leu Val Ser Thr Leu -80-75-70 GCA ATT TCT CTC ATG AGT GCA CAA AGC AAC GAT GCG CTA AAT AGG GAA 517 Ala Ile Ser Leu Met Ser Ala Gln Ser Asn Asp Ala Leu Asn Arg Glu -65-60-55 TTT GAA AGG CAA AAG AAC GAA AAT AAT GCA AAG TTT GAT ACT TAT GTA 565 Phe Glu Arg Gln Lys Asn Glu Asn Asn Ala Lys Phe Asp Thr Tyr Val -50-45-40 GCG AAA GCT TAT GGC TCT AAT ATT GAT AAA AAA AAC CAA AAA GAA GTA 613 Ala Lys Ala Tyr Gly Ser Asn Ile Asp Lys Lys Asn Gln Lys Glu Val -35-30-25 GAT Tr-A TTA AGA AGC AGA TTA GCA GGA TTT AAT TTT GAT GTT CCT TAT 661 Asp Ser Leu Arg Ser Arg Leu Ala Gly Phe Asn Phe Asp Val Pro Tyr -20-15-10 TTT TTA CAA GCT GAA GAT ACT AGA CAA CTA CTC AAT GCA AAT TCT GAC 709 Phe Leu Gln Ala Glu Asp Thr Arg Gln Leu Leu Asn Ala Asn Ser Asp -5 1 5 10 TTA TTA AAT ACT ACA GGA AAT GTG ACA GGA CTT ACC GGC GCT TTC AAT 757 Leu Leu Asn Thr Thr Gly Asn Val Thr Gly Leu Thr Gly Ala Phe Asn 15 20 25 GGT GAG AAT ATC AAA TTT ACA GTA TTT GAT GGT GGT AGA ATT TAT GAA 805 Gly Glu Asn Ile Lys Phe Thr Val Phe Asp Gly Gly Arg Ile Tyr Glu 30 35 40 ACA CAT GAT GCT TTT AAT AAT GCA ACA GGA AGA ATT ACT AAT AAA GAA 853 Thr His Asp Ala Phe Asn Asn Ala Thr Gly Arg Ile Thr Asn Lys Glu 45 50 55 GCT TCG ACA CAA TCT TAC TCT GAT QC TQ AQ GGT GTA GGT AGT TTC 901 Ala Ser Thr Gln Ser Tyr Ser Asp His Ser Thr Gly Val Gly Ser Phe 60 65 70 75 ATT GGT GGA AAA TCA GTA AAC CTT TCA AGT GGA GGA GTT CCA GTT GGA 949 Ile Gly Gly Lys Ser Val Asn Leu Ser Ser Gly Gly Val Pro Val Gly 80 85 90

AAT Gr-A AAA GGT GTC GCT ATA AAT TCA ACA ATG GAT AGC TAT ATG TTT 997 Asn Ala Lys Gly Val Ala Ile Asn Ser Thr Met Asp Ser Tyr Met Phe 95 100 105 AAC ACA ACA Ar-A TTA CCT AAC AAT ACA GCT ACT AGT ACC GTA TTC CAA 1045 Asn Thr Thr Thr Leu Pro Asn Asn Thr Ala Thr Ser Thr Val Phe Gln 110 115 120 AAA ATA TTA ATT GCT CAA CCA AAT ATA TCT AAT CAC TCT TAC GGT GTA 1093 Lys Ile Leu Ile Ala Gln Pro Asn Ile Ser Asn His Ser Tyr Gly Val 125 130 135 AAT TCA GGT TGG ACA GAA ACA TAT GCA AAT AAT GAT TAC GCT TCT TTT 1141 Asn Ser Gly Trp Thr Glu Thr Tyr Ala Asn Asn Asp Tyr Ala Ser Phe 140 145 150 155 ACT TAC AAT GGT TAT AAA GCT GGA GAT ACA TTT TAT GAC TAC CAA GGA 1189 Thr Tyr Asn Gly Tyr Lys Ala Gly Asp Thr Phe Tyr Asp Tyr Gln Gly 160 165 170 ACG TAT AAT ACT AAT GAC GTA AAC TAT GAT AAT ATT GCC TAT AAT AAT 1237 Thr Tyr Asn Thr Asn Asp Val Asn Tyr Asp Asn Ile Ala Tyr Asn Asn 175 180 185 CCT TCA TAT ATT ATT GTA AAG TCA GCA GGT AAC TAT TTT AAT ATG GGA 1285 Pro Ser Tyr Ile Ile Val Lys Ser Ala Gly Asn Tyr Phe Asn Met Gly 190 195 200 CCA ACT GGT ACA GGC TCA TCT GCT CCA AAA TAC TAT AAT AGC GGC AAT 1333 Pro Thr Gly Thr Gly Ser Ser Ala Pro Lys Tyr Tyr Asn Ser Gly Asn 205 210 215 GGA GCT GTT GCT TTT Gr-A GCG ACT GAC ACA CTT CCT CCA AAC AAC TGT 1381 Gly Ala Val Ala Phe Ala Ala Thr Asp Thr Leu Pro Pro Asn Asn Cys 220 225 230 235 TCT TTA GGA TAT GAC TGT ATT GGT ACA GGT TCA TTA GCG AAA AAC ATT 1429 Ser Leu Gly Tyr Asp Cys Ile Gly Thr Gly Ser Leu Ala Lys Asn Ile 240 245 250 ATA GTT GTT GGA GCC ACA GAT ATA ATT ACG ACA AAT AAT AAT CGT TAT 1477 Ile Val Val Gly Ala Thr Asp Ile Ile Thr Thr Asn Asn Asn Arg Tyr 255 260 265 ACC ACA GCA AGC GAT GTT GTT CAC TCA AGC TAT AGC AGT GCA GGA CCA 1525 Thr Thr Ala Ser Asp Val Val His Ser Ser Tyr Ser Ser Ala Gly Pro 270 275 280 AGA GAC GAT GGA GGA ATC AAA CCA GAT ATT TCT ACG GTT GGA ACT AAT 1573 Arg Asp Asp Gly Gly-Ile Lys Pro Asp Ile Ser Thr Val Gly Thr Asn 285 290 295 GTC TTA TAT GCA GCT ACC GTG ACA ACT ACA GGA AGT ACT TGG GCA CAA 1621 Val Leu Tyr Ala Ala Thr Val Thr Thr Thr Gly Ser Thr Trp Ala Gln 300 305 310 315 AAT AGT GGT ACT TCT TTT TCG GCT CCT ATT GTA ACG GGA ATC ATT GGT 1669 Asn Ser Gly Thr Ser Phe SerAla Pro Ile Val Thr Gly Ile Ile Gly 320 325 330

CTA TGG Ar-A CAA ATC AAT AAA CAA TTA TTC AAT AAT GCA CTA TTA AAT 1717 Leu Trp Thr Gln Ile Asn Lys Gln Leu Phe Asn Asn Ala Leu Leu Asn 335 340 345 GCA TCT TCA GCA AAA ACA CTT ACT ATT CAT TCT GCA TCA GAA GCT GGA 1765 Ala Ser Ser Ala Lys Thr Leu Thr Ile His Ser Ala Ser Glu Ala Gly 350 355 360 AAT ATT GGA CCC GAT CCA CAC TTT GGA TGG GGA TTT ATC AAC GCT AAA 1813 Asn Ile Gly Pro Asp Pro His Phe Gly Trp Gly Phe Ile Asn Ala Lys 365 370 375 AAA GGA GCT GAC CTA TTA GTT GGA AAA TCT AAT GGA AGT GTT ATT TTC 1861 Lys Gly Ala Asp Leu Leu Val Gly Lys Ser Asn Gly Ser Val Ile Phe 380 385 390 395 ACT GAT GAA ACT TTA ACA AGT GGA GTT ACT AAC TCT AAA ACA ATA AAA 1909 Thr Asp Glu Thr Leu Thr Ser Gly Val Thr Asn Ser Lys Thr Ile Lys 400 405 410 GCA TCA GGA TCA GAA CCA CTA AAG GTT ACG ATT AGC TGG TTA GAC CCT 1957 Ala Ser Gly Ser Glu Pro Leu Lys Val Thr Ile Ser Trp Leu Asp Pro 415 420 425 AAA TAC ATT CCT AAT TAT CAA TTC GTT TCT GAT GTT TTC AAC AAC CGA 2005 Lys Tyr Ile Pro Asn Tyr Gln Phe Val Ser Asp Val Phe Asn Asn Arg 430 435 440 ACA TCT AAA CTT ATT AAT GAT ATT GAC TTA CGT ATA ATA GAC ACT ACA 2053 Thr Ser Lys Leu Ile Asn Asp Ile Asp Leu Arg Ile Ile Asp Thr Thr 445 450 455 ACA AAC ACA ATT TAT TAT CCA TGG AAA CTT GAT GCA GAT AGT CCT ATG 2101 Thr Asn Thr Ile Tyr Tyr Pro Trp Lys Leu Asp Ala Asp Ser Pro Met 460 465 470 475 ACA CCA GCA ACT AAA GCG GAC AAC ACT GTG GAT AAC GTA GAA CAA GTA 2149 Thr Pro Ala Thr Lys Ala Asp Asn Thr Val Asp Asn Val Glu Gln Val 480 485 490 GTT TTA GAT AAT CCA GTT CCT GGT AGA AAC TAT AGA ATA GAA GTA ACT 2197 Val Leu Asp Asn Pro Val Pro Gly Arg Asn Tyr Arg Ile Glu Val Thr 495 500 505 AAT AAA GGA ACA TTA GTG AAT GAT GCA GGT ACA GCC GCT CCT CAA AAC 2245 Asn Lys Gly Thr Leu Val Asn Asp Ala Gly Thr Ala Ala Pro Gln Asn 510 515 520 TAT TCA ATA ATC GTT ACA GGA TTC AAT GAA GTT TTA GGA ACA AAA GAT 2293 Tyr Ser Ile Ile Val Thr Gly Phe Asn Glu Val Leu Gly Thr Lys Asp 525 530 535 GTT GCA AAT GCA ACA AAT GGA ATT GTT ATT GCA CCA ACC CTT ACA AAA 2341 Val Ala Asn Ala Thr Asn Gly Ile Val Ile Ala Pro Thr Leu Thr Lys 540 545 550 555 GAT GTT GTA AAC ATT TTG AAA GCT CCA AAA AAA TCT AGT TAC ACA GTA 2389 Asp Val Val Asn Ile Leu Lys Ala Pro Lys Lys Ser Ser Tyr Thr Val 560 565 570 TAT GAT TTA TCT GGT AAA AAA TTA CAA AAC GGT GTA ATT AAC AGC GCT 2437

Tyr Asp Leu Ser Gly Lys Lys Leu Gln Asn Gly Val Ile Asn Ser Ala 575 580 585 CAA GAA ACA ATT AGT CTA TCT TCT TAC ACC AAT GGT ATC TAT ATC ATT 2485 Gln Glu Thr Ile Ser Leu Ser Ser Tyr Thr Asn Gly Ile Tyr Ile Ile 590 595 600 GAA GTA AAA ACT GAT AAA GAC GTT ATT TCT AAG AAA GTT ATC AAA GAA 2533 Glu Val Lys Thr Asp Lys Asp Val Ile Ser Lys Lys Val Ile Lys Glu 605 610 615 TAA TTATAATTCA CATATAATTT TACGCAAAAC ACTAACACTT GTTAGTGTTT 2586 * 620 TGTTTTTTTA TGTATATTTG CTTCCTATGG AAAATACACT TCACGAGAAA GTTTCAAAAG 2646 ATATTTTGCT TAAAGCGTAC AATCACATGA TGCTTGCAAA AGCCATGGCA GATATTTATG 2706 AAGAGAACAG AAATGTTACC AAATATGTTC ATAGTACATC AAGAGGTCAT GAAGCAATTC 2766 AACTGGCAAC GGCTTATCAA TTAAAAAAAG AAGATTGGGT TTCTCCTTAT TATAGAGACG 2826 AAAGCATTCT TTTGGGGATA GGTTTTCAGC CTTATCAATT GATGTTGCAG TTATTAGCAA 2886 AAGCTGACGA TCCTTTTTCA GGAGGTAGAT CATACTATTC TCATCCTTCG AGCAGAGATG 2946 AAGACAAACC AAAAATCATT CATCAAAGTT CAGCAACAGG AATGCAGACC ATTCCTACTG 3006 CGGGAGTTGC CCAAGGAATA AAATATATTC AGGATTTTAA TTTACAAGAA TTTGAAAACA 3066 ATCCTGTTGT TGTTTGCAGT CTTGGAGATA ATTCTGTGAC TGAAGGTGAA GTAAGTGAAG 3126 CGTTGCAATT TGCAGCTTTA CACCAGCTTC CTATTATTTT CTTGGTTCAG GATAATGAAT 3186 GGGGAATTTC TGTTACCAAA GAAGAAGCAA GAACTTGTGA TGCTTATGAT TTCGTAGCCG 3246 GATTTGAAGG TCTTGGAAGA ATGCGAGTTG ACGGAACCAA TTTCGTTGAA AGCTTCGAAG 3306 TGATGAAAAA AGCTGTCGAT TTTGTAAGAG CAGAAAGAAA ACCTTTGGTT GTTTGCGCAA 3366 AAACAGTTTT GATTGGTCAC CATACTTCCG GAGTGAGAAG AGAATTC 3413 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 700 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Lys Lys His Leu Leu Leu Val Ser Thr Leu Ala Ile Ser Leu Met -80-75-70-65 Ser Ala Gln Ser Asn Asp Ala Leu Asn Arg Glu Phe Glu Arg Gln Lys -60-55-50

Asn Glu Asn Asn Ala Lys Phe Asp Thr Tyr Val Ala Lys Ala Tyr Gly -45-40-35 Ser Asn Ile Asp Lys Lys Asn Gln Lys Glu Val Asp Ser Leu Arg Ser -30-25-20 Arg Leu Ala Gly Phe Asn Phe Asp Val Pro Tyr Phe Leu Gln Ala Glu -15-10-5 Asp Thr Arg Gln Leu Leu Asn Ala Asn Ser Asp Leu Leu Asn Thr Thr 1 5 10 15 Gly Asn Val Thr Gly Leu Thr Gly Ala Phe Asn Gly Glu Asn Ile Lys 20 25 30 Phe Thr Val Phe Asp Gly Gly Arg Ile Tyr Glu Thr His Asp Ala Phe 35 40 45 Asn Asn Ala Thr Gly Arg Ile Thr Asn Lys Glu Ala Ser Thr Gln Ser 50 55 60 Tyr Ser Asp His Ser Thr Gly Val Gly Ser Phe Ile Gly Gly Lys Ser 65 70 75 80 Val Asn Leu Ser Ser Gly Gly Val Pro Val Gly Asn Ala Lys Gly Val 85 90 95 Ala Ile Asn Ser Thr Met Asp Ser Tyr Met Phe Asn Thr Thr Thr Leu 100 105 110 Pro Asn Asn Thr Ala Thr Ser Thr Val Phe Gln Lys Ile Leu Ile Ala 115 120 125 Gln Pro Asn Ile Ser Asn His Ser Tyr Gly Val Asn Ser Gly Trp Thr 130 135 140 Glu Thr Tyr Ala Asn Asn Asp Tyr Ala Ser Phe Thr Tyr Asn Gly Tyr 145 150 155 160 Lys Ala Gly Asp Thr Phe Tyr Asp Tyr Gln Gly Thr Tyr Asn Thr Asn 165 170 175 Asp Val Asn Tyr Asp Asn Ile Ala Tyr Asn Asn Pro Ser Tyr Ile Ile 180 185 190 Val Lys Ser Ala Gly Asn Tyr Phe Asn Met Gly Pro Thr Gly Thr Gly 195 200 205 Ser Ser Ala Pro Lys Tyr Tyr Asn Ser Gly Asn Gly Ala Val Ala Phe 210 215 220 Ala Ala Thr Asp Thr Leu Pro Pro Asn Asn Cys Ser Leu Gly Tyr Asp 225 230 235 240 Cys Ile Gly Thr Gly Ser Leu Ala Lys Asn Ile Ile Val Val Gly Ala 245 250 255 Thr Asp Ile Ile Thr Thr Asn Asn Asn Arg Tyr Thr Thr Ala Ser Asp 260 265 270 Val Val His Ser Ser Tyr Ser Ser Ala Gly Pro Arg Asp Asp Gly Gly

275 280 285 Ile Lys Pro Asp Ile Ser Thr Val Gly Thr Asn Val Leu Tyr Ala Ala 290 295 300 Thr Val Thr Thr Thr Gly Ser Thr Trp Ala Gln Asn Ser Gly Thr Ser 305 310 315 320 Phe Ser Ala Pro Ile Val Thr Gly Ile Ile Gly Leu Trp Thr Gln Ile 325 330 335 Asn Lys Gln Leu Phe Asn Asn Ala Leu Leu Asn Ala Ser Ser Ala Lys 340 345 350 Thr Leu Thr Ile His Ser Ala Ser Glu Ala Gly Asn Ile Gly Pro Asp 355 360 365 Pro His Phe Gly Trp Gly Phe Ile Asn Ala Lys Lys Gly Ala Asp Leu 370 375 380 Leu Val Gly Lys Ser Asn Gly Ser Val Ile Phe Thr Asp Glu Thr Leu 385 390 395 400 Thr Ser Gly Val Thr Asn Ser Lys Thr Ile Lys Ala Ser Gly Ser Glu 405 410 415 Pro Leu Lys Val Thr Ile Ser Trp Leu Asp Pro Lys Tyr Ile Pro Asn 420 425 430 Tyr Gln Phe Val Ser Asp Val Phe Asn Asn Arg Thr Ser Lys Leu Ile 435 440 445 Asn Asp Ile Asp Leu Arg Ile Ile Asp Thr Thr Thr Asn Thr Ile Tyr 450 455 460 Tyr Pro Trp Lys Leu Asp Ala Asp Ser Pro Met Thr Pro Ala Thr Lys 465 470 475 480 Ala Asp Asn Thr Val Asp Asn Val Glu Gln Val Val Leu Asp Asn Pro 485 490 495 Val Pro Gly Arg Asn Tyr Arg Ile Glu Val Thr Asn Lys Gly Thr Leu 500 505 510 Val Asn Asp Ala Gly Thr Ala Ala Pro Gln Asn Tyr Ser Ile Ile Val 515 520 525 Thr Gly Phe Asn Glu Val Leu Gly Thr Lys Asp Val Ala Asn Ala Thr 530 535 540 Asn Gly Ile Val Ile Ala Pro Thr Leu Thr Lys Asp Val Val Asn Ile 545 550 555 560 Leu Lys Ala Pro Lys Lys Ser Ser Tyr Thr Val Tyr Asp Leu Ser Gly 565 570 575 Lys Lys Leu Gln Asn Gly Val Ile Asn Ser Ala Gln Glu Thr Ile Ser 580 585 590 Leu Ser Ser Tyr Thr Asn Gly Ile Tyr Ile Ile Glu Val Lys Thr Asp 595 600 605 Lys Asp Val Ile Ser Lys Lys Val Ile Lys Glu 610 615