JP2003284553A - Biologically active protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biologically active protein highly stable in-vivo, and usable for efficiently inhibiting topical inflammatory when transplanting cultured skin. <P>SOLUTION: A nucleic acid expressing a fused peptide of a peptide having properties of specifically binding to collagen, with a peptide having activities of an acetylhydrolase of a platelet activating factor is prepared, and a recombinant is prepared by using a vector containing the nucleic acid. The fused protein is isolated from a grown product by growing the recombinant. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein having collagen-binding ability and platelet activating factor acetylhydrolase activity, and a nucleic acid encoding the protein.

[0002]

2. Description of the Related Art When using artificial skin for the treatment of burns, it is necessary to reduce inflammation caused by the antigenicity of artificial skin as much as possible. If no, and if inflammation occurs, voltaren, loxonin, etc. have been used appropriately. However, since these drugs do not stay at the application site of artificial skin for a long period of time, it was necessary to administer these drugs regularly and frequently as long as inflammation occurred.

On the other hand, as fusion proteins of collagen-binding domain of collagenase and other proteins, collagen-binding fibroblast growth factor (CBFGF) and collagen-binding epidermal growth factor (CBEGF) were conventionally known. (Proc. Natl. Acad. USA, 95 (1998), 7018-702
3). However, no attempts have been made so far to introduce a collagen-binding domain into an enzyme protein and maintain the enzyme activity at a specific site.

[0004]

[Problems to be Solved by the Invention] Loxonin and voltaren are not highly stable in vivo, and even if these drugs are used to suppress inflammation in the skin for culture and transplantation, the drugs are bloodstream. As it diffuses from the administration site by riding on, the potency is drastically reduced. Therefore, in order to maintain the function of suppressing the inflammation caused by the transplanted cultured skin, it is necessary to administer these drugs to the patient regularly and frequently, which is very complicated. In addition, by further transporting these drugs in the bloodstream,
Large doses of these agents pose a very dangerous condition to the patient as they affect the immune activity throughout the body. Therefore, there has been a problem that the use of the above-mentioned drug for the purpose of suppressing inflammation at the time of skin transplantation requires careful attention. That is, there is a need for a substance that suppresses inflammation when transplanting cultured skin.

[0005]

Means for Solving the Problems In view of the above-mentioned problems, the present inventors have conducted extensive studies and as a result, have platelets which have binding properties to collagen, which is the main component of skin for culture transplantation, and which have anti-inflammatory action. The present invention has been completed based on the idea of a physiologically active protein having an activator acetylhydrolase activity.

That is, the present invention is as follows. (1) In addition to having the property of binding to collagen,
A physiologically active protein having platelet activating factor acetylhydrolase activity. (2) SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 1
A collagen binding region having the amino acid sequence of 4;
A physiologically active protein comprising the amino acid sequence of SEQ ID NO: 8 or a region comprising the amino acid sequence having one or more amino acid substitutions, deletions, insertions, or transpositions bound thereto. (3) The physiologically active protein according to (2), which has a platelet activating factor acetylhydrolase activity. (4) A nucleic acid encoding the physiologically active protein according to any one of (1) to (3). (5) A DNA encoding the physiologically active protein according to any one of (1) to (3). (6) A vector having the DNA according to (5). (7) SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13
Hybridizes to a DNA consisting of the described nucleotide sequence or a nucleotide sequence complementary thereto under stringent conditions
A vector having in the same read-out region a DNA and a DNA consisting of the nucleotide sequence of SEQ ID NO: 7 or a DNA consisting of a nucleotide sequence complementary thereto under a stringent condition. (8) A recombinant transformed with the vector according to (6) or (7). (9) A method for producing a bioactive protein, which comprises growing the recombinant of (8) and obtaining the bioactive protein from the grown product.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to embodiments of the invention. 1. The Substance of the Present Invention The substance of the present invention is a physiologically active protein having the property of binding to collagen and having the platelet activating factor acetylhydrolase activity.

Examples of the collagen include water-soluble collagen and water-insoluble collagen, and the substance of the present invention preferably has a binding property to water-insoluble collagen. Examples of water-soluble collagen include type II collagen, type III collagen, type IV collagen, type V collagen, type VII collagen, and type VIII collagen, and examples of water insoluble collagen include type I collagen.

The detection and measurement of the property of the substance of the present invention to bind to collagen is exemplified as follows. That is, for example, after mixing the substance of the present invention labeled with radioactivity and collagen, the mixture is subjected to molecular sieve such as electrophoresis and gel filtration, and the radioactivity is detected while detecting the radioactivity with collagen using an anti-collagen antibody, for example. And radioactivity are detected in the same fraction, or water-insoluble collagen is put in a microtube with a filter and measured according to the method described in J. Biol. Chem., 273 (1998), 3643-3648. There is a method.

The platelet activating factor acetylhydrolase activity in the substance of the present invention is an activity possessed by the platelet activating factor acetylhydrolase and selectively hydrolyzes the acetyl group of the phospholipid mediator platelet activating factor (PAF). It is an activity that decomposes and inactivates its physiological effects. This activity is the activity commonly possessed by the enzymes described in the enzyme classification (EC) 3.1.1.47.

Such platelet activating factor acetylhydrolase activity can be measured using, for example, a PAF acetylhydrolase assay kit (Funakoshi). That is, 2-thio-PAF, which is an analog of PAF
Is used as a substrate, and the thiol produced when this substrate is decomposed by the enzymatic activity of PAF-AH is treated with Ellman's reagent (5,
An example is a method of detecting with 5'-dithiobis (2-nitrobenzoic acid)) and measuring the absorbance at 412 nm for quantification.

Such a substance of the present invention has a structure in which the collagen binding site of collagenase and the region having platelet activating factor acetylhydrolase activity are fused. The collagenase may be bacterial or animal, but bacterial collagenase is preferable because it has specificity for binding to water-insoluble collagen. Bacterial collagenase is, for example, Bacillaceae
Gram-positive bacteria such as (Bacillaceae), especially collagenase derived from bacteria of the genus Clostridium ( C. histolyticum , C. perfringens, etc.) are preferably exemplified, but other collagenases may be used as long as they bind to collagen. . Such collagenases include ColH, ColG, Co
Although lA and the like are known, the collagen binding site of any collagenase can be used. Collagen binding site means SEQ ID NO: 10 (ColH), SEQ ID NO: 1
2 (ColG) or the amino acid sequence of SEQ ID NO: 14 (ColA), the number of amino acids is less than 150 and the molecular weight is 30,
The protein having a size of less than 000 Da is referred to, but even if it is a partial sequence, it is applicable to the present invention as long as it has a collagen-binding ability, and is not limited to the above-mentioned specific protein.

These proteins hybridize under stringent conditions to a nucleic acid (DNA) consisting of the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 or a nucleotide sequence complementary to these nucleotide sequences. A protein encoded by a nucleic acid (DNA) having a base sequence of

The term "stringent conditions" as used herein means, for example, 50% formamide, 5 × SSPE (20 ×
SSPE: 2.97 M NaCl, 0.2 M NaH ₂ PO ₄ · H ₂ O, 0.025 M EDTA pH 7.4 aqueous solution), 5 x Denhardt's solution (100 x Denhardt's solution: 1 g Ficoll 400 (Pharmacia), 1
g polyvinylpyrrolidone (PVP-360: Sigma), 1 g
BSA fraction V (bovine serum albumin: Sigma)
Was dissolved in 50 ml of water), incubated in the presence of 0.5% SDS (sodium dodecyl sulfate) at 42 ° C for 16 hours, and then 1 x SSPE containing 0.1% SDS, 0.1 x SSPE containing 0.1% SDS. The conditions under which sequential washing is carried out at 55 ° C. or conditions equivalent to this in the hybridization of nucleic acid are designated. Under such conditions, a DNA consisting of a nucleotide sequence that hybridizes to a certain DNA is 70% of the certain DNA.
It is considered to have the above homology. For example, a DNA consisting of 350 bases has a common base sequence of 245 bases or more, and a DNA consisting of 720 bases has a common base sequence of 500 bases or more. In addition, the DNA sequence consisting of 1200 bases has a common base sequence of 840 bases or more.

The region having platelet activating factor acetylhydrolase activity is a region containing the full length of platelet activating factor acetylhydrolase (PAF-AH) or its active center, and is an amino acid of human secretory (plasma) PAF-AH. Array (Natu
re, 374 (1995), 549-553) and a common amino acid sequence. Specifically, the amino acid sequence of SEQ ID NO: 8 or a substitution or deletion of one or several amino acids therein,
A region having an insertion or a dislocation. The above-mentioned region must have the platelet activating factor acetylhydrolase activity even if it has amino acid substitutions, deletions, insertions or transpositions. It can be easily confirmed by the method described in the examples. Here, the term “a few amino acids” means usually two or more 20
The number is less than or equal to 1, preferably 2 or more and less than 10.

The proteins corresponding to these regions include
A nucleic acid (DNA) consisting of the nucleotide sequence of SEQ ID NO: 7, or
A protein encoded by a nucleic acid (DNA) consisting of a nucleotide sequence that hybridizes to a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 7 under stringent conditions is included.

Specific examples of the substance of the present invention having such a structure include, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 16, a protein consisting of the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence of SEQ ID NO: 20. Proteins are exemplified, but as long as they have a binding property to collagen and have a platelet activating factor acetylhydrolase activity, these amino acid sequences can be used for preparing a protein by a genetic engineering method. Various conventionally used tags may be bound.

2. Method of Preparing Substance of the Present Invention The substance of the present invention can be obtained by a method of preparation comprising the following steps (1) to (4). (1) Construction of a vector in which a collagen-binding region of collagenase is inserted (2) Construction of a vector encoding CBPAF-AH by inserting PAF-AH into the vector of (1) (3) (2) Host cell of vector Preparation of Transformant by Introducing into Transformant (4) (3) Growth of Transformant and Purification of CBPAF-AH from the Developed Product Hereinafter, these steps will be described in detail. (1) Construction of a vector having a collagen-binding region of collagenase inserted The vector having a collagen-binding region inserted, for example, using a known collagenase gene as a template, only the collagen-binding region obtained by amplifying only the collagen-binding region by PCR etc. It can be obtained by a method of inserting a nucleic acid (preferably DNA) encoding the vector into an arbitrary vector according to a conventional method. As for the collagenase gene, if ColH, accession number D29981 (SEQ ID NO: 1) of GenBank, Col
Examples of DNA include GenBank accession number D87215 (SEQ ID NO: 3) and ColA accession number D13791 (SEQ ID NO: 5) for ColA (the amino acid sequence of collagenase encoded by each DNA is SEQ ID NO: 2). (Col
H), SEQ ID NO: 4 (ColG), and SEQ ID NO: 6 (ColA)).

The DNA encoding only the collagen-binding region of ColH is a DNA consisting of the nucleotide sequence of base numbers 3010 to 3366 in SEQ ID NO: 1 (SEQ ID NO: 9), and the DNA encoding only the collagen-binding region of ColG is in SEQ ID NO: 3. Base number 3648 to 435
The DNA consisting of the nucleotide sequence of 8 (SEQ ID NO: 11) and the DNA encoding only the collagen binding region of ColA are the DNA consisting of the nucleotide sequences of 2963 to 3685 of SEQ ID NO: 5 (SEQ ID NO: 1).
3) is applicable, but if the region strictly changes even a part of the above sequence, the binding of the coding region to collagen is not lost. You may have. For example, even a protein in which 25 amino acid residues have been deleted from the N-terminal of each collagen binding region has a binding property to collagen.
In particular, ColG and ColA have two collagen-binding moieties.
It is known to have each of them, and for ColG, base numbers 3648 to 4010 and base numbers 4023 to 43 in SEQ ID NO: 3 are known.
The protein encoded by the nucleotide sequence of 58 also has a collagen-binding ability (J. Biol. Chem., 276 (12), 8761-8770 (200
1)), and for ColA, base number 2963 in SEQ ID NO: 5 ~
It is considered that the protein encoded by 3307 and the nucleotide sequences of nucleotide numbers 3344 to 3685 also has collagen-binding ability. Therefore, only these portions may be used as the collagen binding region.

DNA comprising the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 as the collagen binding region
When using, a primer consisting of the nucleotide sequences of SEQ ID NO: 21 and SEQ ID NO: 22 using SEQ ID NO: 1 as a template,
SEQ ID NO: 23 and SEQ ID NO: 24 using SEQ ID NO: 3 as a template
PCR using the primers consisting of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 5 as a template and the primers consisting of the nucleotide sequences of SEQ ID NO: 25 and SEQ ID NO: 26 to obtain DNAs encoding each collagen binding region as PCR products. You can

The ligation of such a DNA to the vector can be easily performed by a conventional means using a restriction enzyme or a DNA ligase. Vector selection can be routinely and easily performed depending on the type of host cell into which the vector is introduced and transformed and the restriction enzyme used.

(2) Construction of a vector encoding CBPAF-AH by inserting the DNA encoding PAF-AH into the vector (1) The PAF-AH DNA to be inserted into the vector (1) is PAF-AH It is possible to prepare from the cell which expresses. Such cells include, but are not limited to, lymphoid cells, bone marrow cells, thymocytes and the like, and may be cultured cells derived from these cells. Such cells are preferably cells derived from mammals such as humans, monkeys, dogs, cats, horses, cows, pigs, goats, sheep, rats, mice, etc., and particularly cells derived from primates such as humans and monkeys. Are preferred, and those of human origin are most preferred.

For example, when preparing PAF-AH DNA from human-derived U937 cells (cultured cells derived from lymphoma), a cDNA library prepared from total RNA obtained by a conventional method or a 1st strand cDNA is used. Only the DNA of interest can be amplified. In this case, for example, using a sense primer (5 'primer) having the base sequence of SEQ ID NO: 27 and an antisense primer (3' primer) having the base sequence of SEQ ID NO: 28, KO the region consisting of base numbers 1 to 1200
3 ', such as D DNA polymerase (sold by Toyobo Co., Ltd.)
It is possible to perform amplification using an enzyme that does not add poly A to the end. The DNA thus obtained can be easily incorporated into the vector (1) (the vector dephosphorylated after digestion with SmaI) after phosphorylation.

(3) Preparation of Transformant by Introducing Vector of (2) into Host Cell Any host cell can be used as long as the vector can be introduced and the gene can be expressed. It is possible to use as a host cell into which is introduced. For example, when a prokaryotic expression vector is used as a vector, a prokaryotic cell is used as a host cell, and when an expression vector for yeast is used, a yeast is used as a host cell and an expression vector for a mammalian cell is used. When used as the host cell, cells derived from mammals are used as the host cells, and when an expression vector for insect cells is used, the host cells are used as insect cells. The vector of (2) can be introduced into a host cell by a conventional method, and examples thereof include electroporation method and shotgun method.

(4) Growth of transformant of (3) and purification of CBPAF-AH from the grown product The transformant can be grown by a growth method suitable for the transformant. Here, the term "growth" is a concept including not only culturing cells of the transformant but also proliferating and growing the transformant in vivo. CBPAF-AH can be obtained from the grown product thus obtained. The term "grown product" as used herein is a concept that also includes a medium after culturing host cells, a host cell after culturing, and excrements and secretions of a living body when a transformant is grown in a living body.

Isolation and purification of CBPAF-AH from the culture are carried out by affinity purification using an affinity column to which an anti-PAF-AH antibody is bound, and as a vector used for expression, for example, CBPAF-AH is glutathione S-transferase (GST). ),
When a vector that expresses as a fusion protein with His tag, protein A or the like is used, the fusion protein can be easily isolated and purified using a known method suitable for them. When CBPAF-AH is expressed as these fusion proteins, it is also possible to excise only CBPAF-AH from the fusion protein using a known cleavage method. As for such a vector to be used and a method for purifying the obtained protein, those skilled in the art can routinely select and use an appropriate combination.

The substance of the present invention thus prepared is PA
Since it has F-AH activity, it can be used as a platelet activating factor modifier. That is, for example, 24 to 42 ° C
Under the conditions of (preferably 37 ° C.) and pH 4 to 9 (preferably pH 6 to 8), the acetyl group of PAF can be specifically hydrolyzed by allowing the substance of the present invention to coexist with PAF.

[0028]

EXAMPLES The present invention will be described in more detail below with reference to examples. 1. Preparation of Substances of the Present Invention (1) Construction of PAF-AH Incorporating Vector A vector for incorporating PAF-AH was constructed using pGEX-4T-2 manufactured by Pharmacia Biotech. That is, using the primers having the nucleotide sequences of SEQ ID NOs: 21 and 22, DNA of GenBank accession number D29981
DNA encoding the collagen-binding region of ColH prepared by a conventional method using (SEQ ID NO: 1 ColH DNA) as a template
(SEQ ID NO: 9: a blunt end of SmaI at the 5'end and a 3'end
The sticky end of XhoI is located: hCBD). Similarly, a primer having the nucleotide sequences of SEQ ID NOs: 23 and 24 is used, and DNA of GenBank Accession No. D87215 (SEQ ID NO: 3: ColG DNA) is used as a template. Col prepared according to the method
DNA encoding the collagen binding region of G (SEQ ID NO: 1
1: A blunt end of SmaI is arranged at the 5 ′ end and a sticky end of XhoI is arranged at the 3 ′ end: gCBD), SEQ ID NOs: 25 and 26
A DNA encoding a collagen-binding region of ColA (SEQ ID NO: 13: 5 ′) prepared according to a conventional method using a DNA having the accession number D13791 of GenBank (SEQ ID NO: 5: DNA of ColA) as a template using a primer having the described nucleotide sequence. SmaI at the end
Blunt end, and a sticky end of XhoI is placed at the 3'end: aCBD), which was digested with SmaI and XhoI.
A vector was constructed by ligating to EX-4T-2 according to a conventional method. h
The vector introduced with CBD was designated as pCHH, the vector introduced with gCBD was designated as pCHG, and the vector introduced with aCBD was designated as pCPA. In addition, when the gene introduced according to the usual method is expressed, myc
-The vector constructed so that two tags are tandemly ligated into the pCHG digested with BamHI is represented by SEQ ID NO: 29.
The yc-tag gene was introduced and prepared, and this was designated as pCHG (NM2).

(2) Construction of expression vector and transformant Total RNA was prepared from U937 cells (derived from human lymphoma cells) by a conventional method, and the cDNA of PAF-AH was prepared from the 1st strand cDNA.
Was amplified by the PCR method. For the PCR method, DNA having the nucleotide sequence of SEQ ID NO: 27 as the 5'primer and DNA having the nucleotide sequence of SEQ ID NO: 28 as the 3'primer
It was used. This PCR product was phosphorylated by a conventional method and then bound to the SmaI sites of pCHH, pCHG, pCHG (NM2) and pCPA.

Each vector thus obtained was transformed into E.
It was introduced into Escherichia coli DH5α by the electroporation method, 4 clones were separated for each vector, and the nucleotide sequence of the insert was confirmed. Then, a clone in which the nucleotide sequence encoding human plasma secretory PAF-AH (Nature, 374 (1995), 549-553) of nucleotide numbers 1 to 1200 of SEQ ID NO: 7 is inserted in the correct direction is selected, pCHH-PAF-AH
(Derived from pCHH: PAF-AH and the collagen binding region have a nucleotide sequence of SEQ ID NO: 15, and the encoded amino acid sequence of SEQ ID NO: 16.
, PCHG-PAF-AH (derived from pCHG: base sequence of PAF-AH and collagen-binding region is SEQ ID NO: 17, and amino acid sequence encoding is shown in SEQ ID NO: 18), pCPA-PAF-AH (pCPA
Origin: PAF-AH and the collagen binding region have a nucleotide sequence of SEQ ID NO: 19 and the encoded amino acid sequence of SEQ ID NO: 20), and pCHG-NM2-PAF-AH.

The following vector was also prepared for use as a control. That is, the total RNA was prepared from U937 cells (derived from human lymphoma cells) according to a conventional method, and the 1st strand cDNA prepared therefrom was used as a template to prepare an amplification product amplified by the PCR method using SEQ ID NO: 30 and SEQ ID NO: 31 as primers. It was inserted into pCHG (2NM2) by a conventional method. The above-mentioned amplification product contains X at both ends of the DNA encoding PAF-AH.
It has a hoI site, and a stop codon is inserted between the 3'XhoI site and the DNA encoding PAF-AH. Therefore, it is impossible to obtain the substance of the present invention having a collagen binding region even if this vector is expressed. Insert the vector with PAF-AH inserted in the correct orientation into pGEX-N
It was named M2-PAF-AH. pCHG-PAF-AH, pCHG-NM2-PAF-AH, and pGEX-NM2-PAF-AH were introduced into E. coli BL21 by electroporation to obtain transformants.

(3) gCBPAF-AH (pCHG-PAF-AH-derived CBPAF-A
H) and gCB (NM2) PAF-AH (pCHG-NM2-PAF-AH-derived CBPAF-
AH) expression and purification Add 2x YT medium (100 ml), 20% (w / v) glucose aqueous solution (10 ml) and 10 mg / ml ampicillin aqueous solution (1 ml) to a 500 ml flask to prepare a culture medium for transformants. Prepared.
Add 2.2 ml E. coli BL21 solution (100 μl / ml LB medium containing ampicillin to E. coli BL21 and add 37 ° C to the flask).
Overnight culture) was added. The culturing was continued at 37 ° C. until the absorbance at 600 nm (A ₆₀₀ ) was about 0.7 when the culture was measured with an absorptiometer.

When A ₆₀₀ reaches 0.7, 0.1 ml of 0.1M
IPTG (isopropyl-β-D-thiogalactopyranoside)
Was added and the mixture was cultured at 20 ° C. for 20 hours. Then, the culture solution was centrifuged (1000 xg), and 15 to 20 ml of extraction buffer solution (10 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 1 mM P per 100 ml of the culture solution).
It was suspended in MSF (phenylmethylsulfonyl fluoride).

The suspension is sonicated for 10 minutes,
1/9 volume of 10% Triton X-100 solution was added and stirred for 30 minutes at 4 ° C. The suspension was centrifuged at 15,000 xg for 30 minutes. After centrifugation, 3 ml of glutathione per 100 ml of supernatant
-Add Sepharose slurry (50% (v / v) in PBS (Phosphate buffered saline): 1.5 ml as gel) and add 1 at 4 ℃.
Stir for hours. After that, further centrifuge at 2,000 xg for 5 minutes to collect the gel, and use TBS (Tris buffered saline: pH
7.5), 0.03% CHAPS (3-[(3-cholamidopropyl) dimeth
The gel was suspended in ylammonio] -1-propane sulfonate) and packed in a mini column. 10-20 times TBS of gel, 0.03% C
After washing the gel with HAPS, 50 mM Tris-HCl (pH 8.0), 10 mM
The fusion protein was eluted with glutathione solution.

10 units of thrombin (manufactured by Amersham Pharmacia Biotech) was added per 1 mg of the fusion protein, and the mixture was treated at 20 ° C. for 20 hours, and then treated with PBS at 16
After dialysis for an hour, GST was removed using glutathione-sepharose resin to remove GST-free gCBPAF-AH and gCB (NM
2) PAF-AH was obtained.

Sampling was carried out at each stage of the purification, and it was subjected to the method of Laemmli et al. (Nature, 227 (1970), 6) under reducing conditions.
80-685), SDS-PAGE (SDS polyacrylamide gel electrophoresis) was carried out (FIGS. 1 and 2: lanes 1 to 4 in FIG. 1 are E. coli BL21 into which control pGEX-NM2-PAF-AH was introduced. Shows the degree of purification of each sample subjected to the same purification step as described above, lanes 5 to 8 show the degree of purification of gCBPAF-AH, and lanes 1 to 4 of Fig. 2 show the degree of purification of gCB (NM2) PAF-AH. In each figure, lanes 1 and 5 are host cell extracts, lanes 2 and 6 are glutathione-sepharose affinity column flow-through fractions, lanes 3 and 7 are glutathione-sepharose column elution fractions, and lanes 4 and 8 are thrombin-treated. GS
The glutathione-sepharose elution fraction from which T has been removed is shown. ).

Further, the obtained gCBPAF-AH and gCB (NM2) PAF
-AH was subjected to SDS-PAGE (SDS polyacrylamide gel electrophoresis) under reducing conditions, the molecular weight was about 72,400 Da.
(GCBPAF-AH) and 75,300 Da (gCB (NM2) PAF-AH). Furthermore, for gCBPAF-AH after removal of the obtained GST, when the PAF-AH activity was examined using the PAF acetylhydrolase assay kit manufactured by Funakoshi Co., Ltd., an activity equivalent to that of normal secretory PAF-AH was obtained. confirmed.

(4) Preparation of hCBPAF-AH hCBPAF-AH was prepared using pCHH-PAF-AH by the same method as described in (3), and the molecular weight of hCBPAF-AH was measured by SDS under reducing conditions.
-Analysis by PAGE revealed a molecular weight of approximately 76,000 Da. The resulting hCBPAF-AH was examined for PAF-AH activity using a PAF acetylhydrolase assay kit manufactured by Funakoshi Co., Ltd.
The activity equivalent to that of AH was confirmed.

2. GCBPAF-AH and gCB (NM2) PAF-AH (both are GS
Before removing T: GST-gCBPAF-AH and GST-gCB (NM2)
PAF-AH) collagen binding activity was measured by J. Biol. Che.
m., 273 (1998), 3643-3648. That is, GST-gCBPAF-AH or GST-gCB (NM2) PAF-A
H was added to 50 μl of 50 mM Tris · HCl (pH 7.5) containing 5 mM CaCl ₂ , and 15 mg of insoluble type I collagen (manufactured by Sigma) coexisting or not at 25 ° C. for 30 minutes, filter cap The cells were incubated in a microtube with a mark, and then centrifuged. Then, the protein that passed through the filter and did not bind to collagen was removed by SDS-PA.
GE analysis (FIG. 3: lanes 5 and 6 are a control group containing no collagen binding region, lanes 7 and 8 are GST-gCBPAF-A).
H, lanes 9 and 10 are GST-gCB (NM2) PAF-AH, and lanes 5, 7, and 9 are control groups to which collagen is not added. ).
As a result, it was revealed that GST-gCBPAF-AH and GST-gCB (NM2) PAF-AH have binding properties to insoluble type I collagen.

[0040]

Industrial Applicability According to the present invention, there is provided a fusion protein having high stability in vivo and having collagen-binding ability and platelet-activating factor acetylhydrolase activity for efficiently suppressing local inflammation. To be done.

[0041]

[Sequence list]                                SEQUENCE LISTING <110> Seikagaku Corporation <120> Bioactive protein <130> J200103200 <140> <141> <160> 31 <170> PatentIn Ver. 2.1 <210> 1 <211> 3500 <212> DNA <213> Clostridium histolyticum <220> <221> CDS <222> (301) .. (3366) <400> 1 aactcctccc gttttaaata gaatctttat aaatttattt tatcctaata ttctcttata 60 tacttaatta aatattaata aaaaattaat gaacaggtat atcttaacaa aaattaaaca 120 aaaattaaac aaatatataa caaatattaa taaataatgt tgacactact aaaaaatggc 180 gttatacttt aataaaaggc ttatataatt cctcaataca aatattcaga taattatgaa 240 aagagcataa atgaaggaat tatgaatttt ttaaaaatta ttttaaatag ggggaagact 300 atg aaa agg aaa tgt tta tct aaa agg ctt atg tta gct ata aca atg 348 Met Lys Arg Lys Cys Leu Ser Lys Arg Leu Met Leu Ala Ile Thr Met   1 5 10 15 gct aca ata ttt aca gtg aac agt aca tta cca att tat gca gct gta 396 Ala Thr Ile Phe Thr Val Asn Ser Thr Leu Pro Ile Tyr Ala Ala Val              20 25 30 gat aaa aat aat gca aca gca gct gta caa aat gaa agt aag agg tat 444 Asp Lys Asn Asn Ala Thr Ala Ala Val Gln Asn Glu Ser Lys Arg Tyr          35 40 45 aca gta tca tat tta aag act tta aat tat tat gac tta gta gat ttg 492 Thr Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu Val Asp Leu      50 55 60 ctt gtt aag act gaa att gag aat tta cca gac ctt ttt cag tat agt 540 Leu Val Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu Phe Gln Tyr Ser  65 70 75 80 tca gat gca aaa gag ttc tat gga aat aaa act cgt atg agc ttt atc 588 Ser Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr Arg Met Ser Phe Ile                  85 90 95 atg gat gaa att ggt aga agg gca cct cag tat aca gag ata gat cat 636 Met Asp Glu Ile Gly Arg Arg Ala Pro Gln Tyr Thr Glu Ile Asp His             100 105 110 aaa ggt att cct act tta gta gaa gtt gta aga gct gga ttt tac tta 684 Lys Gly Ile Pro Thr Leu Val Glu Val Val Arg Ala Gly Phe Tyr Leu         115 120 125 gga ttc cat aac aag gaa ttg aat gaa ata aac aag agg tct ttt aaa 732 Gly Phe His Asn Lys Glu Leu Asn Glu Ile Asn Lys Arg Ser Phe Lys     130 135 140 gaa agg gta ata cct tct ata tta gca att caa aaa aat cct aat ttt 780 Glu Arg Val Ile Pro Ser Ile Leu Ala Ile Gln Lys Asn Pro Asn Phe 145 150 155 160 aaa cta ggt act gaa gtt caa gat aaa ata gta tct gca aca gga ctt 828 Lys Leu Gly Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr Gly Leu                 165 170 175 tta gct ggt aat gaa aca gcg cct cca gaa gtt gta aat aat ttt aca 876 Leu Ala Gly Asn Glu Thr Ala Pro Pro Glu Val Val Asn Asn Phe Thr             180 185 190 cca ata ctt caa gac tgt ata aag aat ata gac aga tac gct ctt gat 924 Pro Ile Leu Gln Asp Cys Ile Lys Asn Ile Asp Arg Tyr Ala Leu Asp         195 200 205 gat tta aag tca aaa gca tta ttt aat gtt tta gct gca cct acc tat 972 Asp Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala Ala Pro Thr Tyr     210 215 220 gat ata act gag tat tta aga gct act aaa gaa aaa cca gaa aac act 1020 Asp Ile Thr Glu Tyr Leu Arg Ala Thr Lys Glu Lys Pro Glu Asn Thr 225 230 235 240 cct tgg tat ggt aaa ata gat ggg ttt ata aat gaa ctt aaa aag tta 1068 Pro Trp Tyr Gly Lys Ile Asp Gly Phe Ile Asn Glu Leu Lys Lys Leu                 245 250 255 gct ctt tat gga aaa ata aat gat aat aac tct tgg ata ata gat aac 1116 Ala Leu Tyr Gly Lys Ile Asn Asp Asn Asn Ser Trp Ile Ile Asp Asn             260 265 270 ggt ata tat cat ata gca cct tta ggg aag tta cat agc aat aat aaa 1164 Gly Ile Tyr His Ile Ala Pro Leu Gly Lys Leu His Ser Asn Asn Lys         275 280 285 ata gga ata gaa act tta aca gag gtt atg aaa gtt tat cct tat tta 1212 Ile Gly Ile Glu Thr Leu Thr Glu Val Met Lys Val Tyr Pro Tyr Leu     290 295 300 agt atg caa cat tta caa tca gca gat caa att aag cgt cat tat gat 1260 Ser Met Gln His Leu Gln Ser Ala Asp Gln Ile Lys Arg His Tyr Asp 305 310 315 320 tca aaa gat gct gaa gga aac aaa ata cct tta gat aag ttt aaa aag 1308 Ser Lys Asp Ala Glu Gly Asn Lys Ile Pro Leu Asp Lys Phe Lys Lys                 325 330 335 gaa gga aaa gaa aaa tac tgt cca aaa act tat aca ttt gat gat gga 1356 Glu Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr Phe Asp Asp Gly             340 345 350 aaa gta ata ata aaa gct ggt gct aga gta gaa gaa gaa aaa gtt aaa 1404 Lys Val Ile Ile Lys Ala Gly Ala Arg Val Glu Glu Glu Lys Val Lys         355 360 365 aga cta tac tgg gca tca aag gaa gtt aac tct caa ttc ttt aga gta 1452 Arg Leu Tyr Trp Ala Ser Lys Glu Val Asn Ser Gln Phe Phe Arg Val     370 375 380 tac gga ata gac aaa cca tta gaa gaa ggt aat cca gat gat ata tta 1500 Tyr Gly Ile Asp Lys Pro Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu 385 390 395 400 aca atg gtt atc tac aac agt ccc gaa gaa tat aaa ctc aat agt gtt 1548 Thr Met Val Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Ser Val                 405 410 415 cta tac gga tat gat act aat aat ggt ggt atg tat ata gag cca gaa 1596 Leu Tyr Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu Pro Glu             420 425 430 gga act ttc ttc acc tat gaa aga gaa gct caa gaa agc aca tac aca 1644 Gly Thr Phe Phe Thr Tyr Glu Arg Glu Ala Gln Glu Ser Thr Tyr Thr         435 440 445 tta gaa gaa tta ttt aga cat gaa tat aca cat tat ttg caa gga aga 1692 Leu Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Gly Arg     450 455 460 tat gca gtt cca gga caa tgg gga aga aca aaa ctt tat gac aat gat 1740 Tyr Ala Val Pro Gly Gln Trp Gly Arg Thr Lys Leu Tyr Asp Asn Asp 465 470 475 480 aga tta act tgg tat gaa gaa ggt gga gca gaa tta ttt gca ggt tct 1788 Arg Leu Thr Trp Tyr Glu Glu Gly Gly Ala Glu Leu Phe Ala Gly Ser                 485 490 495 act aga act tct gga ata tta cca aga aag agt ata gta tca aat att 1836 Thr Arg Thr Ser Gly Ile Leu Pro Arg Lys Ser Ile Val Ser Asn Ile             500 505 510 cat aat aca aca aga aat aat aga tat aag ctt tca gac act gta cat 1884 His Asn Thr Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr Val His         515 520 525 tct aaa tat ggt gct agt ttt gaa ttc tat aat tat gca tgt atg ttt 1932 Ser Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys Met Phe     530 535 540 atg gat tat atg tat aat aaa gat atg ggt ata tta aat aaa cta aat 1980 Met Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys Leu Asn 545 550 555 560 gat ctt gca aaa aat aat gat gtt gat gga tat gat aat tat att aga 2028 Asp Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr Ile Arg                 565 570 575 gat tta agt tct aat tat gct tta aat gat aaa tat caa gat cat atg 2076 Asp Leu Ser Ser Asn Tyr Ala Leu Asn Asp Lys Tyr Gln Asp His Met             580 585 590 cag gag cgc ata gat aat tat gaa aat tta aca gtg cct ttt gta gct 2124 Gln Glu Arg Ile Asp Asn Tyr Glu Asn Leu Thr Val Pro Phe Val Ala         595 600 605 gat gat tat tta gta agg cat gct tat aag aac cct aat gaa att tat 2172 Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr     610 615 620 tct gaa ata tct gaa gta gca aaa tta aag gat gct aag agt gaa gtt 2220 Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val 625 630 635 640 aag aaa tca caa tat ttt agt acc ttt act ttg aga ggt agt tac aca 2268 Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr                 645 650 655 ggt gga gca tct aag ggg aaa tta gaa gat caa aaa gca atg aat aag 2316 Gly Gly Ala Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys             660 665 670 ttt ata gat gat tca ctt aag aaa tta gat acg tat tct tgg agt ggg 2364 Phe Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp Ser Gly         675 680 685 tat aaa act tta act gct tat ttc act aat tat aaa gtt gac tct tca 2412 Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser     690 695 700 aat aga gtt act tat gat gta gta ttc cac gga tat tta cca aac gaa 2460 Asn Arg Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Asn Glu 705 710 715 720 ggt gat tcc aaa aat tca tta cct tat ggc aag atc aat gga act tac 2508 Gly Asp Ser Lys Asn Ser Leu Pro Tyr Gly Lys Ile Asn Gly Thr Tyr                 725 730 735 aag gga aca gag aaa gaa aaa atc aaa ttc tct agt gaa ggc tct ttc 2556 Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Phe             740 745 750 gat cca gat ggt aaa ata gtt tct tat gaa tgg gat ttc gga gat ggt 2604 Asp Pro Asp Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly Asp Gly         755 760 765 aat aag agt aat gag gaa aat cca gag cat tca tat gac aag gta gga 2652 Asn Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys Val Gly     770 775 780 act tat aca gtg aaa tta aaa gtt act gat gac aag gga gaa tct tca 2700 Thr Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu Ser Ser 785 790 795 800 gta tct act act act gca gaa ata aag gat ctt tca gaa aat aaa ctt 2748 Val Ser Thr Thr Thr Ala Glu Ile Lys Asp Leu Ser Glu Asn Lys Leu                 805 810 815 cca gtt ata tat atg cat gta cct aaa tcc gga gcc tta aat caa aaa 2796 Pro Val Ile Tyr Met His Val Pro Lys Ser Gly Ala Leu Asn Gln Lys             820 825 830 gtt gtt ttc tat gga aaa gga aca tat gac cca gat gga tct atc gca 2844 Val Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala         835 840 845 gga tat caa tgg gac ttt ggt gat gga agt gat ttt agc agt gaa caa 2892 Gly Tyr Gln Trp Asp Phe Gly Asp Gly Ser Asp Phe Ser Ser Glu Gln     850 855 860 aac cca agc cat gta tat act aaa aaa ggt gaa tat act gta aca tta 2940 Asn Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu 865 870 875 880 aga gta atg gat agt agt gga caa atg agt gaa aaa act atg aag att 2988 Arg Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile                 885 890 895 aag att aca gat ccg gta tat cca ata ggc act gaa aaa gaa cca aat 3036 Lys Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn             900 905 910 aac agt aaa gaa act gca agt ggt cca ata gta cca ggt ata cct gtt 3084 Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val         915 920 925 agt gga acc ata gaa aat aca agt gat caa gat tat ttc tat ttt gat 3132 Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp     930 935 940 gtt ata aca cca gga gaa gta aaa ata gat ata aat aaa tta ggg tac 3180 Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr 945 950 955 960 gga gga gct act tgg gta gta tat gat gaa aat aat aat gca gta tct 3228 Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser                 965 970 975 tat gcc act gat gat ggg caa aat tta agt gga aag ttt aag gca gat 3276 Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp             980 985 990 aaa cca ggt aga tat tac atc cat ctt tac atg ttt aat ggt agt tat 3324 Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr         995 1000 1005 atg cca tat aga att aat ata gaa ggt tca gta gga aga taa 3366 Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg    1010 1015 1020 tattttatta gttgaggtaa ctccatataa tagcttagct atttcttatg gagttacttt 3426 ttatatgtaa taaaattttg acttaaatta tgattttttg ctataatggt ttggaaatta 3486 atgatttata attt 3500 <210> 2 <211> 1021 <212> PRT <213> Clostridium histolyticum <400> 2 Met Lys Arg Lys Cys Leu Ser Lys Arg Leu Met Leu Ala Ile Thr Met   1 5 10 15 Ala Thr Ile Phe Thr Val Asn Ser Thr Leu Pro Ile Tyr Ala Ala Val              20 25 30 Asp Lys Asn Asn Ala Thr Ala Ala Val Gln Asn Glu Ser Lys Arg Tyr          35 40 45 Thr Val Ser Tyr Leu Lys Thr Leu Asn Tyr Tyr Asp Leu Val Asp Leu      50 55 60 Leu Val Lys Thr Glu Ile Glu Asn Leu Pro Asp Leu Phe Gln Tyr Ser  65 70 75 80 Ser Asp Ala Lys Glu Phe Tyr Gly Asn Lys Thr Arg Met Ser Phe Ile                  85 90 95 Met Asp Glu Ile Gly Arg Arg Ala Pro Gln Tyr Thr Glu Ile Asp His             100 105 110 Lys Gly Ile Pro Thr Leu Val Glu Val Val Arg Ala Gly Phe Tyr Leu         115 120 125 Gly Phe His Asn Lys Glu Leu Asn Glu Ile Asn Lys Arg Ser Phe Lys     130 135 140 Glu Arg Val Ile Pro Ser Ile Leu Ala Ile Gln Lys Asn Pro Asn Phe 145 150 155 160 Lys Leu Gly Thr Glu Val Gln Asp Lys Ile Val Ser Ala Thr Gly Leu                 165 170 175 Leu Ala Gly Asn Glu Thr Ala Pro Pro Glu Val Val Asn Asn Phe Thr             180 185 190 Pro Ile Leu Gln Asp Cys Ile Lys Asn Ile Asp Arg Tyr Ala Leu Asp         195 200 205 Asp Leu Lys Ser Lys Ala Leu Phe Asn Val Leu Ala Ala Pro Thr Tyr     210 215 220 Asp Ile Thr Glu Tyr Leu Arg Ala Thr Lys Glu Lys Pro Glu Asn Thr 225 230 235 240 Pro Trp Tyr Gly Lys Ile Asp Gly Phe Ile Asn Glu Leu Lys Lys Leu                 245 250 255 Ala Leu Tyr Gly Lys Ile Asn Asp Asn Asn Ser Trp Ile Ile Asp Asn             260 265 270 Gly Ile Tyr His Ile Ala Pro Leu Gly Lys Leu His Ser Asn Asn Lys         275 280 285 Ile Gly Ile Glu Thr Leu Thr Glu Val Met Lys Val Tyr Pro Tyr Leu     290 295 300 Ser Met Gln His Leu Gln Ser Ala Asp Gln Ile Lys Arg His Tyr Asp 305 310 315 320 Ser Lys Asp Ala Glu Gly Asn Lys Ile Pro Leu Asp Lys Phe Lys Lys                 325 330 335 Glu Gly Lys Glu Lys Tyr Cys Pro Lys Thr Tyr Thr Phe Asp Asp Gly             340 345 350 Lys Val Ile Ile Lys Ala Gly Ala Arg Val Glu Glu Glu Lys Val Lys         355 360 365 Arg Leu Tyr Trp Ala Ser Lys Glu Val Asn Ser Gln Phe Phe Arg Val     370 375 380 Tyr Gly Ile Asp Lys Pro Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu 385 390 395 400 Thr Met Val Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Ser Val                 405 410 415 Leu Tyr Gly Tyr Asp Thr Asn Asn Gly Gly Met Tyr Ile Glu Pro Glu             420 425 430 Gly Thr Phe Phe Thr Tyr Glu Arg Glu Ala Gln Glu Ser Thr Tyr Thr         435 440 445 Leu Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Gly Arg     450 455 460 Tyr Ala Val Pro Gly Gln Trp Gly Arg Thr Lys Leu Tyr Asp Asn Asp 465 470 475 480 Arg Leu Thr Trp Tyr Glu Glu Gly Gly Ala Glu Leu Phe Ala Gly Ser                 485 490 495 Thr Arg Thr Ser Gly Ile Leu Pro Arg Lys Ser Ile Val Ser Asn Ile             500 505 510 His Asn Thr Thr Arg Asn Asn Arg Tyr Lys Leu Ser Asp Thr Val His         515 520 525 Ser Lys Tyr Gly Ala Ser Phe Glu Phe Tyr Asn Tyr Ala Cys Met Phe     530 535 540 Met Asp Tyr Met Tyr Asn Lys Asp Met Gly Ile Leu Asn Lys Leu Asn 545 550 555 560 Asp Leu Ala Lys Asn Asn Asp Val Asp Gly Tyr Asp Asn Tyr Ile Arg                 565 570 575 Asp Leu Ser Ser Asn Tyr Ala Leu Asn Asp Lys Tyr Gln Asp His Met             580 585 590 Gln Glu Arg Ile Asp Asn Tyr Glu Asn Leu Thr Val Pro Phe Val Ala         595 600 605 Asp Asp Tyr Leu Val Arg His Ala Tyr Lys Asn Pro Asn Glu Ile Tyr     610 615 620 Ser Glu Ile Ser Glu Val Ala Lys Leu Lys Asp Ala Lys Ser Glu Val 625 630 635 640 Lys Lys Ser Gln Tyr Phe Ser Thr Phe Thr Leu Arg Gly Ser Tyr Thr                 645 650 655 Gly Gly Ala Ser Lys Gly Lys Leu Glu Asp Gln Lys Ala Met Asn Lys             660 665 670 Phe Ile Asp Asp Ser Leu Lys Lys Leu Asp Thr Tyr Ser Trp Ser Gly         675 680 685 Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Lys Val Asp Ser Ser     690 695 700 Asn Arg Val Thr Tyr Asp Val Val Phe His Gly Tyr Leu Pro Asn Glu 705 710 715 720 Gly Asp Ser Lys Asn Ser Leu Pro Tyr Gly Lys Ile Asn Gly Thr Tyr                 725 730 735 Lys Gly Thr Glu Lys Glu Lys Ile Lys Phe Ser Ser Glu Gly Ser Phe             740 745 750 Asp Pro Asp Gly Lys Ile Val Ser Tyr Glu Trp Asp Phe Gly Asp Gly         755 760 765 Asn Lys Ser Asn Glu Glu Asn Pro Glu His Ser Tyr Asp Lys Val Gly     770 775 780 Thr Tyr Thr Val Lys Leu Lys Val Thr Asp Asp Lys Gly Glu Ser Ser 785 790 795 800 Val Ser Thr Thr Thr Ala Glu Ile Lys Asp Leu Ser Glu Asn Lys Leu                 805 810 815 Pro Val Ile Tyr Met His Val Pro Lys Ser Gly Ala Leu Asn Gln Lys             820 825 830 Val Val Phe Tyr Gly Lys Gly Thr Tyr Asp Pro Asp Gly Ser Ile Ala         835 840 845 Gly Tyr Gln Trp Asp Phe Gly Asp Gly Ser Asp Phe Ser Ser Glu Gln     850 855 860 Asn Pro Ser His Val Tyr Thr Lys Lys Gly Glu Tyr Thr Val Thr Leu 865 870 875 880 Arg Val Met Asp Ser Ser Gly Gln Met Ser Glu Lys Thr Met Lys Ile                 885 890 895 Lys Ile Thr Asp Pro Val Tyr Pro Ile Gly Thr Glu Lys Glu Pro Asn             900 905 910 Asn Ser Lys Glu Thr Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val         915 920 925 Ser Gly Thr Ile Glu Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp     930 935 940 Val Ile Thr Pro Gly Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr 945 950 955 960 Gly Gly Ala Thr Trp Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser                 965 970 975 Tyr Ala Thr Asp Asp Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp             980 985 990 Lys Pro Gly Arg Tyr Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr         995 1000 1005 Met Pro Tyr Arg Ile Asn Ile Glu Gly Ser Val Gly Arg    1010 1015 1020 <210> 3 <211> 5914 <212> DNA <213> Clostridium histolyticum <220> <221> CDS <222> (1002) .. (4358) <400> 3 aagcttttat aattctcact atactctcta gatggtataa taagagccca aaatacgaag 60 aatataataa aatataattt aaaaaggatg atttcaatgc gaaaaagagt ttttttgaat 120 atatttttta ttttatgttc ctcaattttt tttatgtcct gcacaggaaa atttcaggtt 180 atagatcggg gtgatggggg agatgaaatt tatttaaaca aacaagatgg tgtgagtttt 240 gagattccta aagtgtggga taaaaattat aagattatca cttctagaga taaaagatat 300 ggcaaaaagt taacttttaa aaaaaaggat aagaaatgca acgttatact tttagaaata 360 tggattttga atgaggaata ttggagtgaa tttaaagatg ttaggaagtt taaacttata 420 ggtaaaagcg aaaaaggcgt agtagtttat tcaagaggta aattagatag catattagaa 480 aataatggat tggacattat gcatcataaa gaagagaaaa agaaagatat agaaaaaatg 540 tacattaaag atgaagaaat tagcgataga atcaaaataa ttagaaatta ataaaaaaat 600 gaaaatagaa aaattcattt tactaaaaat ttatgtttac tttctataac aatctttgta 660 aactgtaaat actaatgtag tattttttag aaaataataa tctgttaaaa agtatattta 720 ggaactaaaa atgaataaat ttataaaaac tatttacaat atctaaaata atgtatataa 780 tttttattaa atagattatt ttggtattaa gggggtgatt gaaagaataa acagaaaatt 840 gatataattc aataaataaa atctaaagag aaatatctaa gtaatacaca aatctaatat 900 taaaaccatt ttaatattaa gaatattttg ttaataggta aaggttaaaa ggcattctat 960 tattaaggtt aaaaggtatt aattattaag ggggattatc t atg aaa aaa aat att 1016                                               Met Lys Lys Asn Ile                                                 1 5 tta aag att ctt atg gat agt tat tct aaa gaa tct aaa att caa act 1064 Leu Lys Ile Leu Met Asp Ser Tyr Ser Lys Glu Ser Lys Ile Gln Thr                  10 15 20 gta cgt agg gtt acg agt gta tca ctt tta gcg gta tat ctt act atg 1112 Val Arg Arg Val Thr Ser Val Ser Leu Leu Ala Val Tyr Leu Thr Met              25 30 35 aat act tca agt tta gtt tta gca aaa cca ata gaa aat act aat gat 1160 Asn Thr Ser Ser Leu Val Leu Ala Lys Pro Ile Glu Asn Thr Asn Asp          40 45 50 act agt ata aaa aat gtg gag aaa tta aga aat gct cca aat gaa gag 1208 Thr Ser Ile Lys Asn Val Glu Lys Leu Arg Asn Ala Pro Asn Glu Glu      55 60 65 aat agt aaa aag gta gaa gat agt aaa aat gat aag gta gaa cat gtg 1256 Asn Ser Lys Lys Val Glu Asp Ser Lys Asn Asp Lys Val Glu His Val  70 75 80 85 aaa aat ata gaa gag gca aag gtt gag caa gtt gca ccc gaa gta aaa 1304 Lys Asn Ile Glu Glu Ala Lys Val Glu Gln Val Ala Pro Glu Val Lys                  90 95 100 tct aaa tca act tta aga agt gct tct ata gcg aat act aat tct gag 1352 Ser Lys Ser Thr Leu Arg Ser Ala Ser Ile Ala Asn Thr Asn Ser Glu             105 110 115 aaa tat gat ttt gag tat tta aat ggt ttg agc tat act gaa ctt aca 1400 Lys Tyr Asp Phe Glu Tyr Leu Asn Gly Leu Ser Tyr Thr Glu Leu Thr         120 125 130 aat tta att aaa aat ata aag tgg aat caa att aat ggt tta ttt aat 1448 Asn Leu Ile Lys Asn Ile Lys Trp Asn Gln Ile Asn Gly Leu Phe Asn     135 140 145 tat agt aca ggt tct caa aag ttc ttt gga gat aaa aat cgt gta caa 1496 Tyr Ser Thr Gly Ser Gln Lys Phe Phe Gly Asp Lys Asn Arg Val Gln 150 155 160 165 gct ata att aat gct tta caa gaa agt gga aga act tac act gca aat 1544 Ala Ile Ile Asn Ala Leu Gln Glu Ser Gly Arg Thr Tyr Thr Ala Asn                 170 175 180 gat atg aag ggt ata gaa act ttc act gag gtt tta aga gct ggt ttt 1592 Asp Met Lys Gly Ile Glu Thr Phe Thr Glu Val Leu Arg Ala Gly Phe             185 190 195 tat tta ggg tac tat aat gat ggt tta tct tat tta aat gat aga aac 1640 Tyr Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr Leu Asn Asp Arg Asn         200 205 210 ttc caa gat aaa tgt ata cct gca atg att gca att caa aaa aat cct 1688 Phe Gln Asp Lys Cys Ile Pro Ala Met Ile Ala Ile Gln Lys Asn Pro     215 220 225 aac ttt aag cta gga act gca gtt caa gat gaa gtt ata act tct tta 1736 Asn Phe Lys Leu Gly Thr Ala Val Gln Asp Glu Val Ile Thr Ser Leu 230 235 240 245 gga aaa cta ata gga aat gct tct gct aat gct gaa gta gtt aat aat 1784 Gly Lys Leu Ile Gly Asn Ala Ser Ala Asn Ala Glu Val Val Asn Asn                 250 255 260 tgt gta cca gtt cta aaa caa ttt aga gaa aac tta aat caa tat gct 1832 Cys Val Pro Val Leu Lys Gln Phe Arg Glu Asn Leu Asn Gln Tyr Ala             265 270 275 cct gat tac gtt aaa gga aca gct gta aat gaa tta att aaa ggt att 1880 Pro Asp Tyr Val Lys Gly Thr Ala Val Asn Glu Leu Ile Lys Gly Ile         280 285 290 gaa ttc gat ttt tct ggt gct gca tat gaa aaa gat gtt aag aca atg 1928 Glu Phe Asp Phe Ser Gly Ala Ala Tyr Glu Lys Asp Val Lys Thr Met     295 300 305 cct tgg tat gga aaa att gat cca ttt ata aat gaa ctt aag gcc tta 1976 Pro Trp Tyr Gly Lys Ile Asp Pro Phe Ile Asn Glu Leu Lys Ala Leu 310 315 320 325 ggt cta tat gga aat ata aca agt gca act gag tgg gca tct gat gtt 2024 Gly Leu Tyr Gly Asn Ile Thr Ser Ala Thr Glu Trp Ala Ser Asp Val                 330 335 340 gga ata tac tat tta agt aaa ttc ggt ctt tac tca act aac cga aat 2072 Gly Ile Tyr Tyr Leu Ser Lys Phe Gly Leu Tyr Ser Thr Asn Arg Asn             345 350 355 gac ata gta cag tca ctt gaa aag gct gta gat atg tat aag tat ggt 2120 Asp Ile Val Gln Ser Leu Glu Lys Ala Val Asp Met Tyr Lys Tyr Gly         360 365 370 aaa ata gcc ttt gta gca atg gag aga ata act tgg gat tat gat ggg 2168 Lys Ile Ala Phe Val Ala Met Glu Arg Ile Thr Trp Asp Tyr Asp Gly     375 380 385 att ggt tct aat ggt aaa aag gtg gat cac gat aag ttc tta gat gat 2216 Ile Gly Ser Asn Gly Lys Lys Val Asp His Asp Lys Phe Leu Asp Asp 390 395 400 405 gct gaa aaa cat tat ctg cca aag aca tat act ttt gat aat gga acc 2264 Ala Glu Lys His Tyr Leu Pro Lys Thr Tyr Thr Phe Asp Asn Gly Thr                 410 415 420 ttt att ata aga gca ggg gat aag gta tcc gaa gaa aaa ata aaa agg 2312 Phe Ile Ile Arg Ala Gly Asp Lys Val Ser Glu Glu Lys Ile Lys Arg             425 430 435 cta tat tgg gca tca aga gaa gtg aag tct caa ttc cat aga gta gtt 2360 Leu Tyr Trp Ala Ser Arg Glu Val Lys Ser Gln Phe His Arg Val Val         440 445 450 ggc aat gat aaa gct tta gag gtg gga aat gcc gat gat gtt tta act 2408 Gly Asn Asp Lys Ala Leu Glu Val Gly Asn Ala Asp Asp Val Leu Thr     455 460 465 atg aaa ata ttt aat agc cca gaa gaa tat aaa ttt aat acc aat ata 2456 Met Lys Ile Phe Asn Ser Pro Glu Glu Tyr Lys Phe Asn Thr Asn Ile 470 475 480 485 aat ggt gta agc act gat aat ggt ggt cta tat ata gaa cca aga ggg 2504 Asn Gly Val Ser Thr Asp Asn Gly Gly Leu Tyr Ile Glu Pro Arg Gly                 490 495 500 act ttc tac act tat gag aga aca cct caa caa agt ata ttt agt ctt 2552 Thr Phe Tyr Thr Tyr Glu Arg Thr Pro Gln Gln Ser Ile Phe Ser Leu             505 510 515 gaa gaa ttg ttt aga cat gaa tat act cac tat tta caa gcg aga tat 2600 Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr Leu Gln Ala Arg Tyr         520 525 530 ctt gta gat ggt tta tgg ggg caa ggt cca ttt tat gaa aaa aat aga 2648 Leu Val Asp Gly Leu Trp Gly Gln Gly Pro Phe Tyr Glu Lys Asn Arg     535 540 545 tta act tgg ttt gat gaa ggt aca gct gaa ttc ttt gca gga tct acc 2696 Leu Thr Trp Phe Asp Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr 550 555 560 565 cgt aca tct ggt gtt tta cca aga aaa tca ata tta gga tat ttg gct 2744 Arg Thr Ser Gly Val Leu Pro Arg Lys Ser Ile Leu Gly Tyr Leu Ala                 570 575 580 aag gat aaa gta gat cat aga tac tca tta aag aag act ctt aat tca 2792 Lys Asp Lys Val Asp His Arg Tyr Ser Leu Lys Lys Thr Leu Asn Ser             585 590 595 ggg tat gat gac agt gat tgg atg ttc tat aat tat gga ttt gca gtt 2840 Gly Tyr Asp Asp Ser Asp Trp Met Phe Tyr Asn Tyr Gly Phe Ala Val         600 605 610 gca cat tac cta tat gaa aaa gat atg cct aca ttt att aag atg aat 2888 Ala His Tyr Leu Tyr Glu Lys Asp Met Pro Thr Phe Ile Lys Met Asn     615 620 625 aaa gct ata ttg aat aca gat gtg aaa tct tat gat gaa ata ata aaa 2936 Lys Ala Ile Leu Asn Thr Asp Val Lys Ser Tyr Asp Glu Ile Ile Lys 630 635 640 645 aaa tta agt gat gat gca aat aaa aat aca gaa tat caa aac cat att 2984 Lys Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu Tyr Gln Asn His Ile                 650 655 660 caa gag tta gca gat aaa tat caa gga gca ggc ata cct cta gta tca 3032 Gln Glu Leu Ala Asp Lys Tyr Gln Gly Ala Gly Ile Pro Leu Val Ser             665 670 675 gat gat tac tta aaa gat cat gga tat aag aaa gca tct gaa gta tat 3080 Asp Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys Ala Ser Glu Val Tyr         680 685 690 tct gaa att tca aaa gct gct tct ctt aca aac act agt gta aca gca 3128 Ser Glu Ile Ser Lys Ala Ala Ser Leu Thr Asn Thr Ser Val Thr Ala     695 700 705 gaa aaa tct caa tat ttt aac aca ttc act tta aga gga act tat aca 3176 Glu Lys Ser Gln Tyr Phe Asn Thr Phe Thr Leu Arg Gly Thr Tyr Thr 710 715 720 725 ggt gaa act tct aaa ggt gaa ttt aaa gat tgg gat gaa atg agt aaa 3224 Gly Glu Thr Ser Lys Gly Glu Phe Lys Asp Trp Asp Glu Met Ser Lys                 730 735 740 aaa tta gat gga act ttg gag tcc ctt gct aaa aat tct tgg agt gga 3272 Lys Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys Asn Ser Trp Ser Gly             745 750 755 tac aaa act tta aca gca tac ttt acg aat tat aga gtt aca agc gat 3320 Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr Arg Val Thr Ser Asp         760 765 770 aat aaa gtt caa tat gat gta gtt ttc cat ggg gtt tta aca gat aat 3368 Asn Lys Val Gln Tyr Asp Val Val Phe His Gly Val Leu Thr Asp Asn     775 780 785 gcg gat att agt aac aat aag gct cca ata gca aag gta act gga cca 3416 Ala Asp Ile Ser Asn Asn Lys Ala Pro Ile Ala Lys Val Thr Gly Pro 790 795 800 805 agc act ggt gct gta gga aga aat att gaa ttt agt gga aaa gat agt 3464 Ser Thr Gly Ala Val Gly Arg Asn Ile Glu Phe Ser Gly Lys Asp Ser                 810 815 820 aaa gat gaa gat ggt aaa ata gta tca tat gat tgg gat ttt ggc gat 3512 Lys Asp Glu Asp Gly Lys Ile Val Ser Tyr Asp Trp Asp Phe Gly Asp             825 830 835 ggt gca act agt aga ggc aaa aat tca gta cat gct tac aaa aaa gca 3560 Gly Ala Thr Ser Arg Gly Lys Asn Ser Val His Ala Tyr Lys Lys Ala         840 845 850 gga aca tat aat gtt aca tta aaa gta act gac gat aag ggt gca aca 3608 Gly Thr Tyr Asn Val Thr Leu Lys Val Thr Asp Asp Lys Gly Ala Thr     855 860 865 gct aca gaa agc ttt act ata gaa ata aag aac gaa gat aca aca aca 3656 Ala Thr Glu Ser Phe Thr Ile Glu Ile Lys Asn Glu Asp Thr Thr Thr 870 875 880 885 cct ata act aaa gaa atg gaa cct aat gat gat ata aaa gag gct aat 3704 Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp Ile Lys Glu Ala Asn                 890 895 900 ggt cca ata gtt gaa ggt gtt act gta aaa ggt gat tta aat ggt tct 3752 Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly Asp Leu Asn Gly Ser             905 910 915 gat gat gct gat acc ttc tat ttt gat gta aaa gaa gat ggt gat gtt 3800 Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp Gly Asp Val         920 925 930 aca att gaa ctt cct tat tca ggg tca tct aat ttc aca tgg tta gtt 3848 Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr Trp Leu Val     935 940 945 tat aaa gag gga gac gat caa aac cat att gca agt ggt ata gat aag 3896 Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala Ser Gly Ile Asp Lys 950 955 960 965 aat aac tca aaa gtt gga aca ttt aaa tct aca aaa gga aga cat tat 3944 Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr Lys Gly Arg His Tyr                 970 975 980 gtg ttt ata tat aaa cac gat tct gct tca aat ata tcc tat tct tta 3992 Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn Ile Ser Tyr Ser Leu             985 990 995 aac ata aaa gga tta ggt aac gag aaa ttg aag gaa aaa gaa aat aat 4040 Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys Glu Asn Asn        1000 1005 1010 gat tct tct gat aaa gct aca gtt ata cca aat ttc aat acc act atg 4088 Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn Phe Asn Thr Thr Met    1015 1020 1025 caa ggt tca ctt tta ggt gat gat tca aga gat tat tat tct ttt gag 4136 Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr Ser Phe Glu 1030 1035 1040 1045 gtt aag gaa gaa ggc gaa gtt aat ata gaa cta gat aaa aag gat gaa 4184 Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu Asp Lys Lys Asp Glu                1050 1055 1060 ttt ggt gta aca tgg aca cta cat cca gag tca aat att aat gac aga 4232 Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser Asn Ile Asn Asp Arg            1065 1070 1075 ata act tac gga caa gtt gat ggt aat aag gta tct aat aaa gtt aaa 4280 Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val Ser Asn Lys Val Lys        1080 1085 1090 tta aga cca gga aaa tat tat cta ctt gtt tat aaa tac tca gga tca 4328 Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr Ser Gly Ser    1095 1100 1105 gga aac tat gag tta agg gta aat aaa taa tttatcttat aaaaaagagt 4378 Gly Asn Tyr Glu Leu Arg Val Asn Lys 1110 1115 gtgcctaata catggcacac tctttttatt tatttttttc ttttaaaaga tctctgattt 4438 caccaagtaa ctcttcttct cttgaaattt caggaatctt agcttcttca actgcttctt 4498 cttttctttt aaatctgttt attagtctta taaataggaa tattgaaaaa gaaattatta 4558 agaagtccaa tatattttgt ataaattgac cataattaag agtcaaaggt ttttctgaat 4618 ttaatccatg aagtgtaagt tttgcgctag taaaattaat tccacctaag ataagtccta 4678 gaataggcat tataacatca tttactaaag atgttacaat ctttccgaag gcaccaccta 4738 tgataacacc tacagcaaga tcgactacat tacctttcat ggcaaattcc ttaaaatctt 4798 tccacataaa aatcctccta aagtatttaa tattaattat taaataacaa gtataatctt 4858 atatttaaat ttaacattaa ttatactaaa tatcaatatg aaattattaa aagttttaca 4918 tttatatgat ataaataata ttggtattta atattatcag gttgattgtt ctttgtgttc 4978 tttaaatttc aaaaaatatg atataatata agagatagta tcgttgtttg atatatctat 5038 ttaaaaaaaa ttacttagtt ttgttaagag gtgttttaaa tgaagattat gtttatatct 5098 gatattcatg gttctttata ttttttaaat aaagcattag aaagatttga agaggaaaaa 5158 gcagattata tagggatttt aggagatgta ttatatcatg gacctagaaa tgatttacca 5218 aaagaatata atccaaagga tgttgcaaaa atcctaaata ggtataaaaa taaaataata 5278 gccgtaaggg gaaattgtga tagtgaagta gatcaaatgc ttatagacta tccaatgctt 5338 agtgattata gtataatttt ttttaataac aagaaaatat ttttaactca cggacatatt 5398 tttaataaag ataatatgcc tcattttaat ataggagata ttatgataag tggtcatact 5458 catattccaa gtatagaaca tatagacggg gtaactttta taaatcctgg ttctatatct 5518 atacctaaag gtggaagtga aaattcttat ggtattttaa atgaggatgg attctcaatt 5578 aaaaatttaa atggaaaggt tatattaact ttaaatatat aatagactaa aggaggaata 5638 agaatgaata caatagaaat ggttttaaat agtttaaaag aggcagggga accgctaaag 5698 gctggagaga tagcagaaaa gactggtatt gacaaaaaag aagtggataa agctataaaa 5758 aaattaaaag ccgaagaaaa gataacttct cctaagaggt gttattatac tattgcataa 5818 tattatctca catgataata ttaaaatata ttaaaaaata ataaaatgta atgtttaggt 5878 tctatttgta gattaatagg gaaagtggtt gaattc 5914 <210> 4 <211> 1118 <212> PRT <213> Clostridium histolyticum <400> 4 Met Lys Lys Asn Ile Leu Lys Ile Leu Met Asp Ser Tyr Ser Lys Glu   1 5 10 15 Ser Lys Ile Gln Thr Val Arg Arg Val Thr Ser Val Ser Leu Leu Ala              20 25 30 Val Tyr Leu Thr Met Asn Thr Ser Ser Leu Val Leu Ala Lys Pro Ile          35 40 45 Glu Asn Thr Asn Asp Thr Ser Ile Lys Asn Val Glu Lys Leu Arg Asn      50 55 60 Ala Pro Asn Glu Glu Asn Ser Lys Lys Val Glu Asp Ser Lys Asn Asp  65 70 75 80 Lys Val Glu His Val Lys Asn Ile Glu Glu Ala Lys Val Glu Gln Val                  85 90 95 Ala Pro Glu Val Lys Ser Lys Ser Thr Leu Arg Ser Ala Ser Ile Ala             100 105 110 Asn Thr Asn Ser Glu Lys Tyr Asp Phe Glu Tyr Leu Asn Gly Leu Ser         115 120 125 Tyr Thr Glu Leu Thr Asn Leu Ile Lys Asn Ile Lys Trp Asn Gln Ile     130 135 140 Asn Gly Leu Phe Asn Tyr Ser Thr Gly Ser Gln Lys Phe Phe Gly Asp 145 150 155 160 Lys Asn Arg Val Gln Ala Ile Ile Asn Ala Leu Gln Glu Ser Gly Arg                 165 170 175 Thr Tyr Thr Ala Asn Asp Met Lys Gly Ile Glu Thr Phe Thr Glu Val             180 185 190 Leu Arg Ala Gly Phe Tyr Leu Gly Tyr Tyr Asn Asp Gly Leu Ser Tyr         195 200 205 Leu Asn Asp Arg Asn Phe Gln Asp Lys Cys Ile Pro Ala Met Ile Ala     210 215 220 Ile Gln Lys Asn Pro Asn Phe Lys Leu Gly Thr Ala Val Gln Asp Glu 225 230 235 240 Val Ile Thr Ser Leu Gly Lys Leu Ile Gly Asn Ala Ser Ala Asn Ala                 245 250 255 Glu Val Val Asn Asn Cys Val Pro Val Leu Lys Gln Phe Arg Glu Asn             260 265 270 Leu Asn Gln Tyr Ala Pro Asp Tyr Val Lys Gly Thr Ala Val Asn Glu         275 280 285 Leu Ile Lys Gly Ile Glu Phe Asp Phe Ser Gly Ala Ala Tyr Glu Lys     290 295 300 Asp Val Lys Thr Met Pro Trp Tyr Gly Lys Ile Asp Pro Phe Ile Asn 305 310 315 320 Glu Leu Lys Ala Leu Gly Leu Tyr Gly Asn Ile Thr Ser Ala Thr Glu                 325 330 335 Trp Ala Ser Asp Val Gly Ile Tyr Tyr Leu Ser Lys Phe Gly Leu Tyr             340 345 350 Ser Thr Asn Arg Asn Asp Ile Val Gln Ser Leu Glu Lys Ala Val Asp         355 360 365 Met Tyr Lys Tyr Gly Lys Ile Ala Phe Val Ala Met Glu Arg Ile Thr     370 375 380 Trp Asp Tyr Asp Gly Ile Gly Ser Asn Gly Lys Lys Val Asp His Asp 385 390 395 400 Lys Phe Leu Asp Asp Ala Glu Lys His Tyr Leu Pro Lys Thr Tyr Thr                 405 410 415 Phe Asp Asn Gly Thr Phe Ile Ile Arg Ala Gly Asp Lys Val Ser Glu             420 425 430 Glu Lys Ile Lys Arg Leu Tyr Trp Ala Ser Arg Glu Val Lys Ser Gln         435 440 445 Phe His Arg Val Val Gly Asn Asp Lys Ala Leu Glu Val Gly Asn Ala     450 455 460 Asp Asp Val Leu Thr Met Lys Ile Phe Asn Ser Pro Glu Glu Tyr Lys 465 470 475 480 Phe Asn Thr Asn Ile Asn Gly Val Ser Thr Asp Asn Gly Gly Leu Tyr                 485 490 495 Ile Glu Pro Arg Gly Thr Phe Tyr Thr Tyr Glu Arg Thr Pro Gln Gln             500 505 510 Ser Ile Phe Ser Leu Glu Glu Leu Phe Arg His Glu Tyr Thr His Tyr         515 520 525 Leu Gln Ala Arg Tyr Leu Val Asp Gly Leu Trp Gly Gln Gly Pro Phe     530 535 540 Tyr Glu Lys Asn Arg Leu Thr Trp Phe Asp Glu Gly Thr Ala Glu Phe 545 550 555 560 Phe Ala Gly Ser Thr Arg Thr Ser Gly Val Leu Pro Arg Lys Ser Ile                 565 570 575 Leu Gly Tyr Leu Ala Lys Asp Lys Val Asp His Arg Tyr Ser Leu Lys             580 585 590 Lys Thr Leu Asn Ser Gly Tyr Asp Asp Ser Asp Trp Met Phe Tyr Asn         595 600 605 Tyr Gly Phe Ala Val Ala His Tyr Leu Tyr Glu Lys Asp Met Pro Thr     610 615 620 Phe Ile Lys Met Asn Lys Ala Ile Leu Asn Thr Asp Val Lys Ser Tyr 625 630 635 640 Asp Glu Ile Ile Lys Lys Leu Ser Asp Asp Ala Asn Lys Asn Thr Glu                 645 650 655 Tyr Gln Asn His Ile Gln Glu Leu Ala Asp Lys Tyr Gln Gly Ala Gly             660 665 670 Ile Pro Leu Val Ser Asp Asp Tyr Leu Lys Asp His Gly Tyr Lys Lys         675 680 685 Ala Ser Glu Val Tyr Ser Glu Ile Ser Lys Ala Ala Ser Leu Thr Asn     690 695 700 Thr Ser Val Thr Ala Glu Lys Ser Gln Tyr Phe Asn Thr Phe Thr Leu 705 710 715 720 Arg Gly Thr Tyr Thr Gly Glu Thr Ser Lys Gly Glu Phe Lys Asp Trp                 725 730 735 Asp Glu Met Ser Lys Lys Leu Asp Gly Thr Leu Glu Ser Leu Ala Lys             740 745 750 Asn Ser Trp Ser Gly Tyr Lys Thr Leu Thr Ala Tyr Phe Thr Asn Tyr         755 760 765 Arg Val Thr Ser Asp Asn Lys Val Gln Tyr Asp Val Val Phe His Gly     770 775 780 Val Leu Thr Asp Asn Ala Asp Ile Ser Asn Asn Lys Ala Pro Ile Ala 785 790 795 800 Lys Val Thr Gly Pro Ser Thr Gly Ala Val Gly Arg Asn Ile Glu Phe                 805 810 815 Ser Gly Lys Asp Ser Lys Asp Glu Asp Gly Lys Ile Val Ser Tyr Asp             820 825 830 Trp Asp Phe Gly Asp Gly Ala Thr Ser Arg Gly Lys Asn Ser Val His         835 840 845 Ala Tyr Lys Lys Ala Gly Thr Tyr Asn Val Thr Leu Lys Val Thr Asp     850 855 860 Asp Lys Gly Ala Thr Ala Thr Glu Ser Phe Thr Ile Glu Ile Lys Asn 865 870 875 880 Glu Asp Thr Thr Thr Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp                 885 890 895 Ile Lys Glu Ala Asn Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly             900 905 910 Asp Leu Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys         915 920 925 Glu Asp Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn     930 935 940 Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala 945 950 955 960 Ser Gly Ile Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr                 965 970 975 Lys Gly Arg His Tyr Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn             980 985 990 Ile Ser Tyr Ser Leu Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys         995 1000 1005 Glu Lys Glu Asn Asn Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn    1010 1015 1020 Phe Asn Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp 1025 1030 1035 1040 Tyr Tyr Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu                1045 1050 1055 Asp Lys Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser            1060 1065 1070 Asn Ile Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val        1075 1080 1085 Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr    1090 1095 1100 Lys Tyr Ser Gly Ser Gly Asn Tyr Glu Leu Arg Val Asn Lys 1105 1110 1115 <210> 5 <211> 4141 <212> DNA <213> Clostridium perfringens <220> <221> CDS <222> (371) .. (3685) <400> 5 aacaattacc taaatacctt tttaatatta cggtgaagaa tgtaatttac tgagttgcca 60 ataaagtaca ttgaatcacc gtaatatttt tttgtgtgat taactggaat aataatacat 120 ttaattttag ttattaatgt attaaaaaaa taactacaaa aaataaaaaa taataggatt 180 atttagaaat gacatataaa aaattacaaa ttttatgtta taattaaaaa gttactaaat 240 taataacaaa tggtgaatga tttttaaatg tgtaaggtta ttttaaatta tgtatataag 300 aaaacttcag cttaaataag caaagtttta tttatataaa tattatttaa gatagaggag 360 gcatttaaac atg aag aaa aac tta aaa agg gga gag cta aca aaa ctg 409            Met Lys Lys Asn Leu Lys Arg Gly Glu Leu Thr Lys Leu              1 5 10 aag tta gtt gaa aga tgg tca gct acc ttt act tta gca gca ttt att 457 Lys Leu Val Glu Arg Trp Ser Ala Thr Phe Thr Leu Ala Ala Phe Ile      15 20 25 tta ttt aat agc tca ttt aaa gta ttt gca gct gat aaa aag gta gag 505 Leu Phe Asn Ser Ser Phe Lys Val Phe Ala Ala Asp Lys Lys Val Glu  30 35 40 45 aat agt aat aat gga cag att act aga gag att aat gct gat cag att 553 Asn Ser Asn Asn Gly Gln Ile Thr Arg Glu Ile Asn Ala Asp Gln Ile                  50 55 60 tct aaa aca gaa tta aat aat gag gta gct aca gac aat aat aga cca 601 Ser Lys Thr Glu Leu Asn Asn Glu Val Ala Thr Asp Asn Asn Arg Pro              65 70 75 tta gga cct agt att gct cca tca aga gca aga aac aac aag atc tat 649 Leu Gly Pro Ser Ile Ala Pro Ser Arg Ala Arg Asn Asn Lys Ile Tyr          80 85 90 aca ttc gat gaa ctt aac aga atg aat tat agt gat cta gtt gaa tta 697 Thr Phe Asp Glu Leu Asn Arg Met Asn Tyr Ser Asp Leu Val Glu Leu      95 100 105 ata aaa aca ata agt tat gaa aac gta cca gac tta ttt aat ttt aat 745 Ile Lys Thr Ile Ser Tyr Glu Asn Val Pro Asp Leu Phe Asn Phe Asn 110 115 120 125 gat ggt tca tat act ttc ttt agt aat aga gat cgt gta caa gct ata 793 Asp Gly Ser Tyr Thr Phe Phe Ser Asn Arg Asp Arg Val Gln Ala Ile                 130 135 140 ata tat ggt cta gag gat agt gga aga act tat aca gca gat gat gat 841 Ile Tyr Gly Leu Glu Asp Ser Gly Arg Thr Tyr Thr Ala Asp Asp Asp             145 150 155 aag gga att cca act tta gtt gag ttt tta aga gct gga tat tat tta 889 Lys Gly Ile Pro Thr Leu Val Glu Phe Leu Arg Ala Gly Tyr Tyr Leu         160 165 170 gga ttt tat aat aaa caa tta tca tac cta aat aca cca cag tta aaa 937 Gly Phe Tyr Asn Lys Gln Leu Ser Tyr Leu Asn Thr Pro Gln Leu Lys     175 180 185 aat gag tgt tta cca gct atg aaa gcg att caa tat aat agt aat ttt 985 Asn Glu Cys Leu Pro Ala Met Lys Ala Ile Gln Tyr Asn Ser Asn Phe 190 195 200 205 aga tta gga aca aag gcg caa gat gga gtt gtt gag gct tta gga aga 1033 Arg Leu Gly Thr Lys Ala Gln Asp Gly Val Val Glu Ala Leu Gly Arg                 210 215 220 ctt ata ggt aat gct tca gca gat cca gaa gtt att aat aat tgc ata 1081 Leu Ile Gly Asn Ala Ser Ala Asp Pro Glu Val Ile Asn Asn Cys Ile             225 230 235 tat gtt tta agt gat ttt aaa gat aat ata gat aag tat ggt tct aac 1129 Tyr Val Leu Ser Asp Phe Lys Asp Asn Ile Asp Lys Tyr Gly Ser Asn         240 245 250 tat agc aag gga aat gca gta ttc aac ctt atg aaa ggt att gat tat 1177 Tyr Ser Lys Gly Asn Ala Val Phe Asn Leu Met Lys Gly Ile Asp Tyr     255 260 265 tat act aat tca gta ata tac aat act aag gga tat gat gct aaa aac 1225 Tyr Thr Asn Ser Val Ile Tyr Asn Thr Lys Gly Tyr Asp Ala Lys Asn 270 275 280 285 act gag ttc tat aat aga ata gat cca tat atg gaa aga tta gaa agt 1273 Thr Glu Phe Tyr Asn Arg Ile Asp Pro Tyr Met Glu Arg Leu Glu Ser                 290 295 300 tta tgt aca ata ggt gat aag tta aat aat gat aat gct tgg ctt gta 1321 Leu Cys Thr Ile Gly Asp Lys Leu Asn Asn Asp Asn Ala Trp Leu Val             305 310 315 aat aat gcc tta tat tac aca ggt aga atg ggt aag ttt aga gaa gac 1369 Asn Asn Ala Leu Tyr Tyr Thr Gly Arg Met Gly Lys Phe Arg Glu Asp         320 325 330 cca tca ata tct caa aga gct tta gaa aga gct atg aag gag tat cct 1417 Pro Ser Ile Ser Gln Arg Ala Leu Glu Arg Ala Met Lys Glu Tyr Pro     335 340 345 tat tta tca tat caa tat att gaa gct gcc aat gat tta gat tta aat 1465 Tyr Leu Ser Tyr Gln Tyr Ile Glu Ala Ala Asn Asp Leu Asp Leu Asn 350 355 360 365 ttt ggt ggc aaa aat tca tca gga aat gat ata gat ttc aat aag ata 1513 Phe Gly Gly Lys Asn Ser Ser Gly Asn Asp Ile Asp Phe Asn Lys Ile                 370 375 380 aaa gca gat gca agg gaa aaa tat ctt cca aaa aca tat act ttt gat 1561 Lys Ala Asp Ala Arg Glu Lys Tyr Leu Pro Lys Thr Tyr Thr Phe Asp             385 390 395 gat ggt aaa ttt gta gta aaa gct ggt gat aaa gta aca gaa gag aag 1609 Asp Gly Lys Phe Val Val Lys Ala Gly Asp Lys Val Thr Glu Glu Lys         400 405 410 ata aaa aga tta tat tgg gct tca aag gaa gtt aag gct caa ttc atg 1657 Ile Lys Arg Leu Tyr Trp Ala Ser Lys Glu Val Lys Ala Gln Phe Met     415 420 425 aga gta gtt caa aat gat aag gct tta gaa gag gga aat cca gat gat 1705 Arg Val Val Gln Asn Asp Lys Ala Leu Glu Glu Gly Asn Pro Asp Asp 430 435 440 445 att tta act gtt gtt att tat aac tca cca gaa gag tat aag tta aat 1753 Ile Leu Thr Val Val Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn                 450 455 460 cgt ata ata aat gga ttt agt act gat aat ggt ggt ata tat att gaa 1801 Arg Ile Ile Asn Gly Phe Ser Thr Asp Asn Gly Gly Ile Tyr Ile Glu             465 470 475 aac ata gga act ttc ttt act tat gaa aga aca cca gag gaa agt ata 1849 Asn Ile Gly Thr Phe Phe Thr Tyr Glu Arg Thr Pro Glu Glu Ser Ile         480 485 490 tat aca tta gaa gaa tta ttc cgt cat gaa ttt act cac tat ctt caa 1897 Tyr Thr Leu Glu Glu Leu Phe Arg His Glu Phe Thr His Tyr Leu Gln     495 500 505 ggt aga tat gta gtt cct gga atg tgg ggg caa gga gaa ttc tat caa 1945 Gly Arg Tyr Val Val Gly Met Trp Gly Gln Gly Glu Phe Tyr Gln 510 515 520 525 gag gga gtt tta act tgg tat gaa gaa gga aca gca gag ttc ttt gca 1993 Glu Gly Val Leu Thr Trp Tyr Glu Glu Gly Thr Ala Glu Phe Phe Ala                 530 535 540 ggt tca act aga act gat gga ata aaa cca aga aaa tca gtt aca caa 2041 Gly Ser Thr Arg Thr Asp Gly Ile Lys Pro Arg Lys Ser Val Thr Gln             545 550 555 ggg tta gct tac gat aga aat aat aga atg tct tta tat ggt gta tta 2089 Gly Leu Ala Tyr Asp Arg Asn Asn Arg Met Ser Leu Tyr Gly Val Leu         560 565 570 cat gct aaa tat ggc tca tgg gat ttc tat aat tat gga ttt gct cta 2137 His Ala Lys Tyr Gly Ser Trp Asp Phe Tyr Asn Tyr Gly Phe Ala Leu     575 580 585 tca aac tac atg tac aac aat aac atg gga atg ttt aat aag atg aca 2185 Ser Asn Tyr Met Tyr Asn Asn Asn Met Gly Met Phe Asn Lys Met Thr 590 595 600 605 aat tac ata aag aat aat gat gta tct ggt tat aaa gat tat att gca 2233 Asn Tyr Ile Lys Asn Asn Asp Val Ser Gly Tyr Lys Asp Tyr Ile Ala                 610 615 620 tca atg agt agt gat tac gga tta aat gat aaa tat caa gac tat atg 2281 Ser Met Ser Ser Asp Tyr Gly Leu Asn Asp Lys Tyr Gln Asp Tyr Met             625 630 635 gat tct tta tta aac aat att gat aac tta gat gtt cct tta gtt tca 2329 Asp Ser Leu Leu Asn Asn Ile Asp Asn Leu Asp Val Pro Leu Val Ser         640 645 650 gat gag tat gta aat gga cat gaa gct aag gat ata aat gaa ata act 2377 Asp Glu Tyr Val Asn Gly His Glu Ala Lys Asp Ile Asn Glu Ile Thr     655 660 665 aat gac ata aaa gaa gtt tca aat ata aaa gat ctt tct agt aat gtt 2425 Asn Asp Ile Lys Glu Val Ser Asn Ile Lys Asp Leu Ser Ser Asn Val 670 675 680 685 gaa aag tct caa ttc ttt act act tac gat atg aga gga aca tat gta 2473 Glu Lys Ser Gln Phe Phe Thr Thr Tyr Asp Met Arg Gly Thr Tyr Val                 690 695 700 ggg gga aga agt caa ggg gaa gaa aat gac tgg aaa gat atg aat tct 2521 Gly Gly Arg Ser Gln Gly Glu Glu Asn Asp Trp Lys Asp Met Asn Ser             705 710 715 aag tta aat gat atg tta aaa gaa tta tct aaa aag agc tgg aat ggt 2569 Lys Leu Asn Asp Met Leu Lys Glu Leu Ser Lys Lys Ser Trp Asn Gly         720 725 730 tat aaa act gtt act gca tac ttt gta aac cat aaa gta gat gaa aat 2617 Tyr Lys Thr Val Thr Ala Tyr Phe Val Asn His Lys Val Asp Glu Asn     735 740 745 ggt aac tat gtt tat gat gtt gta ttc cat gga atg aat aca gat aca 2665 Gly Asn Tyr Val Tyr Asp Val Val Phe His Gly Met Asn Thr Asp Thr 750 755 760 765 aat act gat gtt cat gtt aat aaa gag cct aag gct gtt ata aaa tct 2713 Asn Thr Asp Val His Val Asn Lys Glu Pro Lys Ala Val Ile Lys Ser                 770 775 780 gat tct tca gta ata gtt gaa gaa gaa att aat ttt gat gga aca gag 2761 Asp Ser Ser Val Ile Val Glu Glu Glu Ile Asn Phe Asp Gly Thr Glu             785 790 795 tca aag gat gaa gat ggt gaa atc aaa gct tat gaa tgg gac ttt gga 2809 Ser Lys Asp Glu Asp Gly Glu Ile Lys Ala Tyr Glu Trp Asp Phe Gly         800 805 810 gat gga gaa aaa tct aat gag gct aaa gcc act cat aag tat aat aaa 2857 Asp Gly Glu Lys Ser Asn Glu Ala Lys Ala Thr His Lys Tyr Asn Lys     815 820 825 act gga gaa tat gaa gta aaa tta aca gtt aca gat aat aac ggt gga 2905 Thr Gly Glu Tyr Glu Val Lys Leu Thr Val Thr Asp Asn Asn Gly Gly 830 835 840 845 ata aat act gaa agt aaa aag ata aaa gta gta gaa gat aaa cct gtt 2953 Ile Asn Thr Glu Ser Lys Lys Ile Lys Val Val Glu Asp Lys Pro Val                 850 855 860 gaa gtt ata aat gaa agc gag cca aac aat gat ttt gaa aag gct aac 3001 Glu Val Ile Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn             865 870 875 caa ata gct aaa tct aat atg tta gtt aag ggt act tta tca gaa gag 3049 Gln Ile Ala Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu         880 885 890 gat tat tca gat aaa tat tat ttt gat gta gct aaa aaa ggc aat gtt 3097 Asp Tyr Ser Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val     895 900 905 aaa atc act ctt aat aat tta aat tca gta gga ata act tgg aca ctt 3145 Lys Ile Thr Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu 910 915 920 925 tat aaa gag gga gac cta aac aat tat gtt tta tat gca act gga aat 3193 Tyr Lys Glu Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn                 930 935 940 gat gga aca gaa tta aag ggt gaa aag act tta gag cct gga aga tac 3241 Asp Gly Thr Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr             945 950 955 tac tta agt gta tat act tat gat aat caa tca gga gct tac aca gta 3289 Tyr Leu Ser Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val         960 965 970 aat gta aaa gga aac ctt aaa aat gaa gtt aaa gaa aca gaa aag gat 3337 Asn Val Lys Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp     975 980 985 gct ata aaa gaa gtt gaa aat aac aat gat ttt gat aaa gct atg aag 3385 Ala Ile Lys Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys 990 995 1000 1005 gta gac agt aat agc aaa ata gtt gga aca tta agc aat gat gat ctt 3433 Val Asp Ser Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu                1010 1015 1020 aag gat att tat agc ata gat ata caa aat cca agt gac tta aac ata 3481 Lys Asp Ile Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile            1025 1030 1035 gta gtt gaa aac tta gat aat ata aaa atg aac tgg tta tta tat tca 3529 Val Val Glu Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser        1040 1045 1050 gct gat gat tta agt aac tat gtg gat tac gct aat gca gat gga aat 3577 Ala Asp Asp Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn    1055 1060 1065 aaa tta agt aac act tgt aag tta aat cca ggt aaa tat tac tta tgt 3625 Lys Leu Ser Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys 1070 1075 1080 1085 gtt tat caa ttt gaa aac tca ggt act gga aat tac aca gta aac tta 3673 Val Tyr Gln Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu                1090 1095 1100 caa aac aaa taa aacaatggtt aaaatagtat aaatagaggc tgtatcaaag 3725 Gln Asn Lys            1105 ctcaaatata gaatttataa acaatacttt tataatttct atgttttaag agggatgata 3785 cagccttaaa ttttctttaa aataagtttt atgtattttt atgaagacta attagtagct 3845 tcttctaaaa cagatgtaca atgaggacat ctagtagctt ttatgtctat tttagtatag 3905 caataaggac aatctttttc tgtagcttca actttaactt cttctttttt cttagtgaag 3965 tttttaagct tgtttatttg ttttataact ataaatatag aaaatgctat tattaagaag 4025 ttaattaagt tatttaaaaa tagtccgtaa tttatagtag cagcaccagc agcttgagct 4085 tcttgtaatg agttaaattg ttgtccactt aagtttatgt ataagttaga gaagtc 4141 <210> 6 <211> 1104 <212> PRT <213> Clostridium perfringens <400> 6 Met Lys Lys Asn Leu Lys Arg Gly Glu Leu Thr Lys Leu Lys Leu Val   1 5 10 15 Glu Arg Trp Ser Ala Thr Phe Thr Leu Ala Ala Phe Ile Leu Phe Asn              20 25 30 Ser Ser Phe Lys Val Phe Ala Ala Asp Lys Lys Val Glu Asn Ser Asn          35 40 45 Asn Gly Gln Ile Thr Arg Glu Ile Asn Ala Asp Gln Ile Ser Lys Thr      50 55 60 Glu Leu Asn Asn Glu Val Ala Thr Asp Asn Asn Arg Pro Leu Gly Pro  65 70 75 80 Ser Ile Ala Pro Ser Arg Ala Arg Asn Asn Lys Ile Tyr Thr Phe Asp                  85 90 95 Glu Leu Asn Arg Met Asn Tyr Ser Asp Leu Val Glu Leu Ile Lys Thr             100 105 110 Ile Ser Tyr Glu Asn Val Pro Asp Leu Phe Asn Phe Asn Asp Gly Ser         115 120 125 Tyr Thr Phe Phe Ser Asn Arg Asp Arg Val Gln Ala Ile Ile Tyr Gly     130 135 140 Leu Glu Asp Ser Gly Arg Thr Tyr Thr Ala Asp Asp Asp Lys Gly Ile 145 150 155 160 Pro Thr Leu Val Glu Phe Leu Arg Ala Gly Tyr Tyr Leu Gly Phe Tyr                 165 170 175 Asn Lys Gln Leu Ser Tyr Leu Asn Thr Pro Gln Leu Lys Asn Glu Cys             180 185 190 Leu Pro Ala Met Lys Ala Ile Gln Tyr Asn Ser Asn Phe Arg Leu Gly         195 200 205 Thr Lys Ala Gln Asp Gly Val Val Glu Ala Leu Gly Arg Leu Ile Gly     210 215 220 Asn Ala Ser Ala Asp Pro Glu Val Ile Asn Asn Cys Ile Tyr Val Leu 225 230 235 240 Ser Asp Phe Lys Asp Asn Ile Asp Lys Tyr Gly Ser Asn Tyr Ser Lys                 245 250 255 Gly Asn Ala Val Phe Asn Leu Met Lys Gly Ile Asp Tyr Tyr Thr Asn             260 265 270 Ser Val Ile Tyr Asn Thr Lys Gly Tyr Asp Ala Lys Asn Thr Glu Phe         275 280 285 Tyr Asn Arg Ile Asp Pro Tyr Met Glu Arg Leu Glu Ser Leu Cys Thr     290 295 300 Ile Gly Asp Lys Leu Asn Asn Asp Asn Ala Trp Leu Val Asn Asn Ala 305 310 315 320 Leu Tyr Tyr Thr Gly Arg Met Gly Lys Phe Arg Glu Asp Pro Ser Ile                 325 330 335 Ser Gln Arg Ala Leu Glu Arg Ala Met Lys Glu Tyr Pro Tyr Leu Ser             340 345 350 Tyr Gln Tyr Ile Glu Ala Ala Asn Asp Leu Asp Leu Asn Phe Gly Gly         355 360 365 Lys Asn Ser Ser Gly Asn Asp Ile Asp Phe Asn Lys Ile Lys Ala Asp     370 375 380 Ala Arg Glu Lys Tyr Leu Pro Lys Thr Tyr Thr Phe Asp Asp Gly Lys 385 390 395 400 Phe Val Val Lys Ala Gly Asp Lys Val Thr Glu Glu Lys Ile Lys Arg                 405 410 415 Leu Tyr Trp Ala Ser Lys Glu Val Lys Ala Gln Phe Met Arg Val Val             420 425 430 Gln Asn Asp Lys Ala Leu Glu Glu Gly Asn Pro Asp Asp Ile Leu Thr         435 440 445 Val Val Ile Tyr Asn Ser Pro Glu Glu Tyr Lys Leu Asn Arg Ile Ile     450 455 460 Asn Gly Phe Ser Thr Asp Asn Gly Gly Ile Tyr Ile Glu Asn Ile Gly 465 470 475 480 Thr Phe Phe Thr Tyr Glu Arg Thr Pro Glu Glu Ser Ile Tyr Thr Leu                 485 490 495 Glu Glu Leu Phe Arg His Glu Phe Thr His Tyr Leu Gln Gly Arg Tyr             500 505 510 Val Val Pro Gly Met Trp Gly Gln Gly Glu Phe Tyr Gln Glu Gly Val         515 520 525 Leu Thr Trp Tyr Glu Glu Gly Thr Ala Glu Phe Phe Ala Gly Ser Thr     530 535 540 Arg Thr Asp Gly Ile Lys Pro Arg Lys Ser Val Thr Gln Gly Leu Ala 545 550 555 560 Tyr Asp Arg Asn Asn Arg Met Ser Leu Tyr Gly Val Leu His Ala Lys                 565 570 575 Tyr Gly Ser Trp Asp Phe Tyr Asn Tyr Gly Phe Ala Leu Ser Asn Tyr             580 585 590 Met Tyr Asn Asn Asn Met Gly Met Phe Asn Lys Met Thr Asn Tyr Ile         595 600 605 Lys Asn Asn Asp Val Ser Gly Tyr Lys Asp Tyr Ile Ala Ser Met Ser     610 615 620 Ser Asp Tyr Gly Leu Asn Asp Lys Tyr Gln Asp Tyr Met Asp Ser Leu 625 630 635 640 Leu Asn Asn Ile Asp Asn Leu Asp Val Pro Leu Val Ser Asp Glu Tyr                 645 650 655 Val Asn Gly His Glu Ala Lys Asp Ile Asn Glu Ile Thr Asn Asp Ile             660 665 670 Lys Glu Val Ser Asn Ile Lys Asp Leu Ser Ser Asn Val Glu Lys Ser         675 680 685 Gln Phe Phe Thr Thr Tyr Asp Met Arg Gly Thr Tyr Val Gly Gly Arg     690 695 700 Ser Gln Gly Glu Glu Asn Asp Trp Lys Asp Met Asn Ser Lys Leu Asn 705 710 715 720 Asp Met Leu Lys Glu Leu Ser Lys Lys Ser Trp Asn Gly Tyr Lys Thr                 725 730 735 Val Thr Ala Tyr Phe Val Asn His Lys Val Asp Glu Asn Gly Asn Tyr             740 745 750 Val Tyr Asp Val Val Phe His Gly Met Asn Thr Asp Thr Asn Thr Asp         755 760 765 Val His Val Asn Lys Glu Pro Lys Ala Val Ile Lys Ser Asp Ser Ser     770 775 780 Val Ile Val Glu Glu Glu Ile Asn Phe Asp Gly Thr Glu Ser Lys Asp 785 790 795 800 Glu Asp Gly Glu Ile Lys Ala Tyr Glu Trp Asp Phe Gly Asp Gly Glu                 805 810 815 Lys Ser Asn Glu Ala Lys Ala Thr His Lys Tyr Asn Lys Thr Gly Glu             820 825 830 Tyr Glu Val Lys Leu Thr Val Thr Asp Asn Asn Gly Gly Ile Asn Thr         835 840 845 Glu Ser Lys Lys Ile Lys Val Val Glu Asp Lys Pro Val Glu Val Ile     850 855 860 Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn Gln Ile Ala 865 870 875 880 Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu Asp Tyr Ser                 885 890 895 Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val Lys Ile Thr             900 905 910 Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu Tyr Lys Glu         915 920 925 Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn Asp Gly Thr     930 935 940 Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr Tyr Leu Ser 945 950 955 960 Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val Asn Val Lys                 965 970 975 Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp Ala Ile Lys             980 985 990 Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys Val Asp Ser         995 1000 1005 Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu Lys Asp Ile    1010 1015 1020 Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile Val Val Glu 1025 1030 1035 1040 Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser Ala Asp Asp                1045 1050 1055 Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn Lys Leu Ser            1060 1065 1070 Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys Val Tyr Gln        1075 1080 1085 Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu Gln Asn Lys    1090 1095 1100 <210> 7 <211> 1203 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (1203) <400> 7 ata caa gta ctg atg gct gct gca agc ttt ggc caa act aaa atc ccc 48 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 cgg gga aat ggg cct tat tcc gtt ggt tgt aca gac tta atg ttt gat 96 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 cac act aat aag ggc acc ttc ttg cgt tta tat tat cca tcc caa gat 144 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 aat gat cgc ctt gac acc ctt tgg atc cca aat aaa gaa tat ttt tgg 192 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 ggt ctt agc aaa ttt ctt gga aca cac tgg ctt atg ggc aac att ttg 240 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 agg tta ctc ttt ggt tca atg aca act cct gca aac tgg aat tcc cct 288 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 ctg agg cct ggt gaa aaa tat cca ctt gtt gtt ttt tct cat ggt ctt 336 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 ggg gca ttc agg aca ctt tat tct gct att ggc att gac ctg gca tct 384 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 cat ggg ttt ata gtt gct gct gta gaa cac aga gat aga tct gca tct 432 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 gca act tac tat ttc aag gac caa tct gct gca gaa ata ggg gac aag 480 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 tct tgg ctc tac ctt aga acc ctg aaa caa gag gag gag aca cat ata 528 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 cga aat gag cag gta cgg caa aga gca aaa gaa tgt tcc caa gct ctc 576 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 agt ctg att ctt gac att gat cat gga aag cca gtg aag aat gca tta 624 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 gat tta aag ttt gat atg gaa caa ctg aag gac tct att gat agg gaa 672 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 aaa ata gca gta att gga cat tct ttt ggt gga gca acg gtt att cag 720 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 act ctt agt gaa gat cag aga ttc aga tgt ggt att gcc ctg gat gca 768 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 tgg atg ttt cca ctg ggt gat gaa gta tat tcc aga att cct cag ccc 816 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 ctc ttt ttt atc aac tct gaa tat ttc caa tat cct gct aat atc ata 864 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 aaa atg aaa aaa tgc tac tca cct gat aaa gaa aga aag atg att aca 912 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 atc agg ggt tca gtc cac cag aat ttt gct gac ttc act ttt gca act 960 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 ggc aaa ata att gga cac atg ctc aaa tta aag gga gac ata gat tca 1008 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 aat gta gct att gat ctt agc aac aaa gct tca tta gca ttc tta caa 1056 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 aag cat tta gga ctt cat aaa gat ttt gat cag tgg gac tgc ttg att 1104 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 gaa gga gat gat gag aat ctt att cca ggg acc aac att aac aca acc 1152 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 aat caa cac atc atg tta cag aac tct tca gga ata gag aaa tac aat 1200 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 tag 1203     <210> 8 <211> 400 <212> PRT <213> Homo sapiens <400> 8 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 <210> 9 <211> 357 <212> DNA <213> Clostridium histolyticum <220> <221> CDS <222> (1) .. (357) <400> 9 cca ata ggc act gaa aaa gaa cca aat aac agt aaa gaa act gca agt 48 Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser   1 5 10 15 ggt cca ata gta cca ggt ata cct gtt agt gga acc ata gaa aat aca 96 Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr              20 25 30 agt gat caa gat tat ttc tat ttt gat gtt ata aca cca gga gaa gta 144 Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly Glu Val          35 40 45 aaa ata gat ata aat aaa tta ggg tac gga gga gct act tgg gta gta 192 Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val      50 55 60 tat gat gaa aat aat aat gca gta tct tat gcc act gat gat ggg caa 240 Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln  65 70 75 80 aat tta agt gga aag ttt aag gca gat aaa cca ggt aga tat tac atc 288 Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile                  85 90 95 cat ctt tac atg ttt aat ggt agt tat atg cca tat aga att aat ata 336 His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile             100 105 110 gaa ggt tca gta gga aga taa 357 Glu Gly Ser Val Gly Arg         115 <210> 10 <211> 118 <212> PRT <213> Clostridium histolyticum <400> 10 Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr Ala Ser   1 5 10 15 Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu Asn Thr              20 25 30 Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly Glu Val          35 40 45 Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp Val Val      50 55 60 Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp Gly Gln  65 70 75 80 Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr Tyr Ile                  85 90 95 His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile Asn Ile             100 105 110 Glu Gly Ser Val Gly Arg         115 <210> 11 <211> 711 <212> DNA <213> Clostridium histolyticum <220> <221> CDS <222> (1) .. (711) <400> 11 aca aca aca cct ata act aaa gaa atg gaa cct aat gat gat ata aaa 48 Thr Thr Thr Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp Ile Lys   1 5 10 15 gag gct aat ggt cca ata gtt gaa ggt gtt act gta aaa ggt gat tta 96 Glu Ala Asn Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly Asp Leu              20 25 30 aat ggt tct gat gat gct gat acc ttc tat ttt gat gta aaa gaa gat 144 Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp          35 40 45 ggt gat gtt aca att gaa ctt cct tat tca ggg tca tct aat ttc aca 192 Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr      50 55 60 tgg tta gtt tat aaa gag gga gac gat caa aac cat att gca agt ggt 240 Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala Ser Gly  65 70 75 80 ata gat aag aat aac tca aaa gtt gga aca ttt aaa tct aca aaa gga 288 Ile Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr Lys Gly                  85 90 95 aga cat tat gtg ttt ata tat aaa cac gat tct gct tca aat ata tcc 336 Arg His Tyr Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn Ile Ser             100 105 110 tat tct tta aac ata aaa gga tta ggt aac gag aaa ttg aag gaa aaa 384 Tyr Ser Leu Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys         115 120 125 gaa aat aat gat tct tct gat aaa gct aca gtt ata cca aat ttc aat 432 Glu Asn Asn Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn Phe Asn     130 135 140 acc act atg caa ggt tca ctt tta ggt gat gat tca aga gat tat tat 480 Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr 145 150 155 160 tct ttt gag gtt aag gaa gaa ggc gaa gtt aat ata gaa cta gat aaa 528 Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu Asp Lys                 165 170 175 aag gat gaa ttt ggt gta aca tgg aca cta cat cca gag tca aat att 576 Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser Asn Ile             180 185 190 aat gac aga ata act tac gga caa gtt gat ggt aat aag gta tct aat 624 Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val Ser Asn         195 200 205 aaa gtt aaa tta aga cca gga aaa tat tat cta ctt gtt tat aaa tac 672 Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr     210 215 220 tca gga tca gga aac tat gag tta agg gta aat aaa taa 711 Ser Gly Ser Gly Asn Tyr Glu Leu Arg Val Asn Lys 225 230 235 <210> 12 <211> 236 <212> PRT <213> Clostridium histolyticum <400> 12 Thr Thr Thr Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp Ile Lys   1 5 10 15 Glu Ala Asn Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly Asp Leu              20 25 30 Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys Glu Asp          35 40 45 Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn Phe Thr      50 55 60 Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala Ser Gly  65 70 75 80 Ile Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr Lys Gly                  85 90 95 Arg His Tyr Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn Ile Ser             100 105 110 Tyr Ser Leu Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys Glu Lys         115 120 125 Glu Asn Asn Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn Phe Asn     130 135 140 Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp Tyr Tyr 145 150 155 160 Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu Asp Lys                 165 170 175 Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser Asn Ile             180 185 190 Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val Ser Asn         195 200 205 Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr Lys Tyr     210 215 220 Ser Gly Ser Gly Asn Tyr Glu Leu Arg Val Asn Lys 225 230 235 <210> 13 <211> 723 <212> DNA <213> Clostridium perfringens <220> <221> CDS <222> (1) .. (723) <400> 13 aat gaa agc gag cca aac aat gat ttt gaa aag gct aac caa ata gct 48 Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn Gln Ile Ala   1 5 10 15 aaa tct aat atg tta gtt aag ggt act tta tca gaa gag gat tat tca 96 Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu Asp Tyr Ser              20 25 30 gat aaa tat tat ttt gat gta gct aaa aaa ggc aat gtt aaa atc act 144 Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val Lys Ile Thr          35 40 45 ctt aat aat tta aat tca gta gga ata act tgg aca ctt tat aaa gag 192 Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu Tyr Lys Glu      50 55 60 gga gac cta aac aat tat gtt tta tat gca act gga aat gat gga aca 240 Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn Asp Gly Thr  65 70 75 80 gaa tta aag ggt gaa aag act tta gag cct gga aga tac tac tta agt 288 Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr Tyr Leu Ser                  85 90 95 gta tat act tat gat aat caa tca gga gct tac aca gta aat gta aaa 336 Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val Asn Val Lys             100 105 110 gga aac ctt aaa aat gaa gtt aaa gaa aca gaa aag gat gct ata aaa 384 Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp Ala Ile Lys         115 120 125 gaa gtt gaa aat aac aat gat ttt gat aaa gct atg aag gta gac agt 432 Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys Val Asp Ser     130 135 140 aat agc aaa ata gtt gga aca tta agc aat gat gat ctt aag gat att 480 Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu Lys Asp Ile 145 150 155 160 tat agc ata gat ata caa aat cca agt gac tta aac ata gta gtt gaa 528 Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile Val Val Glu                 165 170 175 aac tta gat aat ata aaa atg aac tgg tta tta tat tca gct gat gat 576 Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser Ala Asp Asp             180 185 190 tta agt aac tat gtg gat tac gct aat gca gat gga aat aaa tta agt 624 Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn Lys Leu Ser         195 200 205 aac act tgt aag tta aat cca ggt aaa tat tac tta tgt gtt tat caa 672 Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys Val Tyr Gln     210 215 220 ttt gaa aac tca ggt act gga aat tac aca gta aac tta caa aac aaa 720 Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu Gln Asn Lys 225 230 235 240 taa 723     <210> 14 <211> 240 <212> PRT <213> Clostridium perfringens <400> 14 Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn Gln Ile Ala   1 5 10 15 Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu Asp Tyr Ser              20 25 30 Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val Lys Ile Thr          35 40 45 Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu Tyr Lys Glu      50 55 60 Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn Asp Gly Thr  65 70 75 80 Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr Tyr Leu Ser                  85 90 95 Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val Asn Val Lys             100 105 110 Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp Ala Ile Lys         115 120 125 Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys Val Asp Ser     130 135 140 Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu Lys Asp Ile 145 150 155 160 Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile Val Val Glu                 165 170 175 Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser Ala Asp Asp             180 185 190 Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn Lys Leu Ser         195 200 205 Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys Val Tyr Gln     210 215 220 Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu Gln Asn Lys 225 230 235 240 <210> 15 <211> 1563 <212> DNA <213> Artificial Sequence <220> <221> CDS <222> (1) .. (1563) <220> <223> Description of Artificial Sequence: DNA cording       hCBPAF-AH <400> 15 ata caa gta ctg atg gct gct gca agc ttt ggc caa act aaa atc ccc 48 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 cgg gga aat ggg cct tat tcc gtt ggt tgt aca gac tta atg ttt gat 96 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 cac act aat aag ggc acc ttc ttg cgt tta tat tat cca tcc caa gat 144 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 aat gat cgc ctt gac acc ctt tgg atc cca aat aaa gaa tat ttt tgg 192 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 ggt ctt agc aaa ttt ctt gga aca cac tgg ctt atg ggc aac att ttg 240 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 agg tta ctc ttt ggt tca atg aca act cct gca aac tgg aat tcc cct 288 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 ctg agg cct ggt gaa aaa tat cca ctt gtt gtt ttt tct cat ggt ctt 336 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 ggg gca ttc agg aca ctt tat tct gct att ggc att gac ctg gca tct 384 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 cat ggg ttt ata gtt gct gct gta gaa cac aga gat aga tct gca tct 432 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 gca act tac tat ttc aag gac caa tct gct gca gaa ata ggg gac aag 480 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 tct tgg ctc tac ctt aga acc ctg aaa caa gag gag gag aca cat ata 528 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 cga aat gag cag gta cgg caa aga gca aaa gaa tgt tcc caa gct ctc 576 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 agt ctg att ctt gac att gat cat gga aag cca gtg aag aat gca tta 624 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 gat tta aag ttt gat atg gaa caa ctg aag gac tct att gat agg gaa 672 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 aaa ata gca gta att gga cat tct ttt ggt gga gca acg gtt att cag 720 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 act ctt agt gaa gat cag aga ttc aga tgt ggt att gcc ctg gat gca 768 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 tgg atg ttt cca ctg ggt gat gaa gta tat tcc aga att cct cag ccc 816 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 ctc ttt ttt atc aac tct gaa tat ttc caa tat cct gct aat atc ata 864 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 aaa atg aaa aaa tgc tac tca cct gat aaa gaa aga aag atg att aca 912 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 atc agg ggt tca gtc cac cag aat ttt gct gac ttc act ttt gca act 960 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 ggc aaa ata att gga cac atg ctc aaa tta aag gga gac ata gat tca 1008 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 aat gta gct att gat ctt agc aac aaa gct tca tta gca ttc tta caa 1056 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 aag cat tta gga ctt cat aaa gat ttt gat cag tgg gac tgc ttg att 1104 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 gaa gga gat gat gag aat ctt att cca ggg acc aac att aac aca acc 1152 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 aat caa cac atc atg tta cag aac tct tca gga ata gag aaa tac aat 1200 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 ccc ggg cca ata ggc act gaa aaa gaa cca aat aac agt aaa gaa act 1248 Pro Gly Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr                 405 410 415 gca agt ggt cca ata gta cca ggt ata cct gtt agt gga acc ata gaa 1296 Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu             420 425 430 aat aca agt gat caa gat tat ttc tat ttt gat gtt ata aca cca gga 1344 Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly         435 440 445 gaa gta aaa ata gat ata aat aaa tta ggg tac gga gga gct act tgg 1392 Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp     450 455 460 gta gta tat gat gaa aat aat aat gca gta tct tat gcc act gat gat 1440 Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp 465 470 475 480 ggg caa aat tta agt gga aag ttt aag gca gat aaa cca ggt aga tat 1488 Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr                 485 490 495 tac atc cat ctt tac atg ttt aat ggt agt tat atg cca tat aga att 1536 Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile             500 505 510 aat ata gaa ggt tca gta gga aga taa 1563 Asn Ile Glu Gly Ser Val Gly Arg         515 520 <210> 16 <211> 520 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: DNA cording       hCBPAF-AH <400> 16 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 Pro Gly Pro Ile Gly Thr Glu Lys Glu Pro Asn Asn Ser Lys Glu Thr                 405 410 415 Ala Ser Gly Pro Ile Val Pro Gly Ile Pro Val Ser Gly Thr Ile Glu             420 425 430 Asn Thr Ser Asp Gln Asp Tyr Phe Tyr Phe Asp Val Ile Thr Pro Gly         435 440 445 Glu Val Lys Ile Asp Ile Asn Lys Leu Gly Tyr Gly Gly Ala Thr Trp     450 455 460 Val Val Tyr Asp Glu Asn Asn Asn Ala Val Ser Tyr Ala Thr Asp Asp 465 470 475 480 Gly Gln Asn Leu Ser Gly Lys Phe Lys Ala Asp Lys Pro Gly Arg Tyr                 485 490 495 Tyr Ile His Leu Tyr Met Phe Asn Gly Ser Tyr Met Pro Tyr Arg Ile             500 505 510 Asn Ile Glu Gly Ser Val Gly Arg         515 520 <210> 17 <211> 1917 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA cording       gCBPAF-AH <220> <221> CDS <222> (1) .. (1917) <400> 17 ata caa gta ctg atg gct gct gca agc ttt ggc caa act aaa atc ccc 48 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 cgg gga aat ggg cct tat tcc gtt ggt tgt aca gac tta atg ttt gat 96 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 cac act aat aag ggc acc ttc ttg cgt tta tat tat cca tcc caa gat 144 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 aat gat cgc ctt gac acc ctt tgg atc cca aat aaa gaa tat ttt tgg 192 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 ggt ctt agc aaa ttt ctt gga aca cac tgg ctt atg ggc aac att ttg 240 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 agg tta ctc ttt ggt tca atg aca act cct gca aac tgg aat tcc cct 288 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 ctg agg cct ggt gaa aaa tat cca ctt gtt gtt ttt tct cat ggt ctt 336 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 ggg gca ttc agg aca ctt tat tct gct att ggc att gac ctg gca tct 384 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 cat ggg ttt ata gtt gct gct gta gaa cac aga gat aga tct gca tct 432 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 gca act tac tat ttc aag gac caa tct gct gca gaa ata ggg gac aag 480 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 tct tgg ctc tac ctt aga acc ctg aaa caa gag gag gag aca cat ata 528 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 cga aat gag cag gta cgg caa aga gca aaa gaa tgt tcc caa gct ctc 576 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 agt ctg att ctt gac att gat cat gga aag cca gtg aag aat gca tta 624 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 gat tta aag ttt gat atg gaa caa ctg aag gac tct att gat agg gaa 672 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 aaa ata gca gta att gga cat tct ttt ggt gga gca acg gtt att cag 720 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 act ctt agt gaa gat cag aga ttc aga tgt ggt att gcc ctg gat gca 768 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 tgg atg ttt cca ctg ggt gat gaa gta tat tcc aga att cct cag ccc 816 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 ctc ttt ttt atc aac tct gaa tat ttc caa tat cct gct aat atc ata 864 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 aaa atg aaa aaa tgc tac tca cct gat aaa gaa aga aag atg att aca 912 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 atc agg ggt tca gtc cac cag aat ttt gct gac ttc act ttt gca act 960 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 ggc aaa ata att gga cac atg ctc aaa tta aag gga gac ata gat tca 1008 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 aat gta gct att gat ctt agc aac aaa gct tca tta gca ttc tta caa 1056 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 aag cat tta gga ctt cat aaa gat ttt gat cag tgg gac tgc ttg att 1104 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 gaa gga gat gat gag aat ctt att cca ggg acc aac att aac aca acc 1152 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 aat caa cac atc atg tta cag aac tct tca gga ata gag aaa tac aat 1200 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 ccc ggg aca aca aca cct ata act aaa gaa atg gaa cct aat gat gat 1248 Pro Gly Thr Thr Thr Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp                 405 410 415 ata aaa gag gct aat ggt cca ata gtt gaa ggt gtt act gta aaa ggt 1296 Ile Lys Glu Ala Asn Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly             420 425 430 gat tta aat ggt tct gat gat gct gat acc ttc tat ttt gat gta aaa 1344 Asp Leu Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys         435 440 445 gaa gat ggt gat gtt aca att gaa ctt cct tat tca ggg tca tct aat 1392 Glu Asp Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn     450 455 460 ttc aca tgg tta gtt tat aaa gag gga gac gat caa aac cat att gca 1440 Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala 465 470 475 480 agt ggt ata gat aag aat aac tca aaa gtt gga aca ttt aaa tct aca 1488 Ser Gly Ile Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr                 485 490 495 aaa gga aga cat tat gtg ttt ata tat aaa cac gat tct gct tca aat 1536 Lys Gly Arg His Tyr Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn             500 505 510 ata tcc tat tct tta aac ata aaa gga tta ggt aac gag aaa ttg aag 1584 Ile Ser Tyr Ser Leu Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys         515 520 525 gaa aaa gaa aat aat gat tct tct gat aaa gct aca gtt ata cca aat 1632 Glu Lys Glu Asn Asn Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn     530 535 540 ttc aat acc act atg caa ggt tca ctt tta ggt gat gat tca aga gat 1680 Phe Asn Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp 545 550 555 560 tat tat tct ttt gag gtt aag gaa gaa ggc gaa gtt aat ata gaa cta 1728 Tyr Tyr Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu                 565 570 575 gat aaa aag gat gaa ttt ggt gta aca tgg aca cta cat cca gag tca 1776 Asp Lys Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser             580 585 590 aat att aat gac aga ata act tac gga caa gtt gat ggt aat aag gta 1824 Asn Ile Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val         595 600 605 tct aat aaa gtt aaa tta aga cca gga aaa tat tat cta ctt gtt tat 1872 Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr     610 615 620 aaa tac tca gga tca gga aac tat gag tta agg gta aat aaa taa 1917 Lys Tyr Ser Gly Ser Gly Asn Tyr Glu Leu Arg Val Asn Lys 625 630 635 <210> 18 <211> 638 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: DNA cording       gCBPAF-AH <400> 18 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 Pro Gly Thr Thr Thr Pro Ile Thr Lys Glu Met Glu Pro Asn Asp Asp                 405 410 415 Ile Lys Glu Ala Asn Gly Pro Ile Val Glu Gly Val Thr Val Lys Gly             420 425 430 Asp Leu Asn Gly Ser Asp Asp Ala Asp Thr Phe Tyr Phe Asp Val Lys         435 440 445 Glu Asp Gly Asp Val Thr Ile Glu Leu Pro Tyr Ser Gly Ser Ser Asn     450 455 460 Phe Thr Trp Leu Val Tyr Lys Glu Gly Asp Asp Gln Asn His Ile Ala 465 470 475 480 Ser Gly Ile Asp Lys Asn Asn Ser Lys Val Gly Thr Phe Lys Ser Thr                 485 490 495 Lys Gly Arg His Tyr Val Phe Ile Tyr Lys His Asp Ser Ala Ser Asn             500 505 510 Ile Ser Tyr Ser Leu Asn Ile Lys Gly Leu Gly Asn Glu Lys Leu Lys         515 520 525 Glu Lys Glu Asn Asn Asp Ser Ser Asp Lys Ala Thr Val Ile Pro Asn     530 535 540 Phe Asn Thr Thr Met Gln Gly Ser Leu Leu Gly Asp Asp Ser Arg Asp 545 550 555 560 Tyr Tyr Ser Phe Glu Val Lys Glu Glu Gly Glu Val Asn Ile Glu Leu                 565 570 575 Asp Lys Lys Asp Glu Phe Gly Val Thr Trp Thr Leu His Pro Glu Ser             580 585 590 Asn Ile Asn Asp Arg Ile Thr Tyr Gly Gln Val Asp Gly Asn Lys Val         595 600 605 Ser Asn Lys Val Lys Leu Arg Pro Gly Lys Tyr Tyr Leu Leu Val Tyr     610 615 620 Lys Tyr Ser Gly Ser Gly Asn Tyr Glu Leu Arg Val Asn Lys 625 630 635 <210> 19 <211> 1929 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA cording       aCBPAF-AH <220> <221> CDS <222> (1) .. (1929) <400> 19 ata caa gta ctg atg gct gct gca agc ttt ggc caa act aaa atc ccc 48 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 cgg gga aat ggg cct tat tcc gtt ggt tgt aca gac tta atg ttt gat 96 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 cac act aat aag ggc acc ttc ttg cgt tta tat tat cca tcc caa gat 144 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 aat gat cgc ctt gac acc ctt tgg atc cca aat aaa gaa tat ttt tgg 192 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 ggt ctt agc aaa ttt ctt gga aca cac tgg ctt atg ggc aac att ttg 240 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 agg tta ctc ttt ggt tca atg aca act cct gca aac tgg aat tcc cct 288 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 ctg agg cct ggt gaa aaa tat cca ctt gtt gtt ttt tct cat ggt ctt 336 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 ggg gca ttc agg aca ctt tat tct gct att ggc att gac ctg gca tct 384 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 cat ggg ttt ata gtt gct gct gta gaa cac aga gat aga tct gca tct 432 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 gca act tac tat ttc aag gac caa tct gct gca gaa ata ggg gac aag 480 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 tct tgg ctc tac ctt aga acc ctg aaa caa gag gag gag aca cat ata 528 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 cga aat gag cag gta cgg caa aga gca aaa gaa tgt tcc caa gct ctc 576 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 agt ctg att ctt gac att gat cat gga aag cca gtg aag aat gca tta 624 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 gat tta aag ttt gat atg gaa caa ctg aag gac tct att gat agg gaa 672 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 aaa ata gca gta att gga cat tct ttt ggt gga gca acg gtt att cag 720 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 act ctt agt gaa gat cag aga ttc aga tgt ggt att gcc ctg gat gca 768 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 tgg atg ttt cca ctg ggt gat gaa gta tat tcc aga att cct cag ccc 816 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 ctc ttt ttt atc aac tct gaa tat ttc caa tat cct gct aat atc ata 864 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 aaa atg aaa aaa tgc tac tca cct gat aaa gaa aga aag atg att aca 912 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 atc agg ggt tca gtc cac cag aat ttt gct gac ttc act ttt gca act 960 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 ggc aaa ata att gga cac atg ctc aaa tta aag gga gac ata gat tca 1008 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 aat gta gct att gat ctt agc aac aaa gct tca tta gca ttc tta caa 1056 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 aag cat tta gga ctt cat aaa gat ttt gat cag tgg gac tgc ttg att 1104 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 gaa gga gat gat gag aat ctt att cca ggg acc aac att aac aca acc 1152 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 aat caa cac atc atg tta cag aac tct tca gga ata gag aaa tac aat 1200 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 ccc ggg aat gaa agc gag cca aac aat gat ttt gaa aag gct aac caa 1248 Pro Gly Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn Gln                 405 410 415 ata gct aaa tct aat atg tta gtt aag ggt act tta tca gaa gag gat 1296 Ile Ala Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu Asp             420 425 430 tat tca gat aaa tat tat ttt gat gta gct aaa aaa ggc aat gtt aaa 1344 Tyr Ser Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val Lys         435 440 445 atc act ctt aat aat tta aat tca gta gga ata act tgg aca ctt tat 1392 Ile Thr Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu Tyr     450 455 460 aaa gag gga gac cta aac aat tat gtt tta tat gca act gga aat gat 1440 Lys Glu Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn Asp 465 470 475 480 gga aca gaa tta aag ggt gaa aag act tta gag cct gga aga tac tac 1488 Gly Thr Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr Tyr                 485 490 495 tta agt gta tat act tat gat aat caa tca gga gct tac aca gta aat 1536 Leu Ser Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val Asn             500 505 510 gta aaa gga aac ctt aaa aat gaa gtt aaa gaa aca gaa aag gat gct 1584 Val Lys Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp Ala         515 520 525 ata aaa gaa gtt gaa aat aac aat gat ttt gat aaa gct atg aag gta 1632 Ile Lys Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys Val     530 535 540 gac agt aat agc aaa ata gtt gga aca tta agc aat gat gat ctt aag 1680 Asp Ser Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu Lys 545 550 555 560 gat att tat agc ata gat ata caa aat cca agt gac tta aac ata gta 1728 Asp Ile Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile Val                 565 570 575 gtt gaa aac tta gat aat ata aaa atg aac tgg tta tta tat tca gct 1776 Val Glu Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser Ala             580 585 590 gat gat tta agt aac tat gtg gat tac gct aat gca gat gga aat aaa 1824 Asp Asp Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn Lys         595 600 605 tta agt aac act tgt aag tta aat cca ggt aaa tat tac tta tgt gtt 1872 Leu Ser Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys Val     610 615 620 tat caa ttt gaa aac tca ggt act gga aat tac aca gta aac tta caa 1920 Tyr Gln Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu Gln 625 630 635 640 aac aaa taa 1929 Asn Lys <210> 20 <211> 642 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: DNA cording       aCBPAF-AH <400> 20 Ile Gln Val Leu Met Ala Ala Ala Ser Phe Gly Gln Thr Lys Ile Pro   1 5 10 15 Arg Gly Asn Gly Pro Tyr Ser Val Gly Cys Thr Asp Leu Met Phe Asp              20 25 30 His Thr Asn Lys Gly Thr Phe Leu Arg Leu Tyr Tyr Pro Ser Gln Asp          35 40 45 Asn Asp Arg Leu Asp Thr Leu Trp Ile Pro Asn Lys Glu Tyr Phe Trp      50 55 60 Gly Leu Ser Lys Phe Leu Gly Thr His Trp Leu Met Gly Asn Ile Leu  65 70 75 80 Arg Leu Leu Phe Gly Ser Met Thr Pro Ala Asn Trp Asn Ser Pro                  85 90 95 Leu Arg Pro Gly Glu Lys Tyr Pro Leu Val Val Phe Ser His Gly Leu             100 105 110 Gly Ala Phe Arg Thr Leu Tyr Ser Ala Ile Gly Ile Asp Leu Ala Ser         115 120 125 His Gly Phe Ile Val Ala Ala Val Glu His Arg Asp Arg Ser Ala Ser     130 135 140 Ala Thr Tyr Tyr Phe Lys Asp Gln Ser Ala Ala Glu Ile Gly Asp Lys 145 150 155 160 Ser Trp Leu Tyr Leu Arg Thr Leu Lys Gln Glu Glu Glu Thr His Ile                 165 170 175 Arg Asn Glu Gln Val Arg Gln Arg Ala Lys Glu Cys Ser Gln Ala Leu             180 185 190 Ser Leu Ile Leu Asp Ile Asp His Gly Lys Pro Val Lys Asn Ala Leu         195 200 205 Asp Leu Lys Phe Asp Met Glu Gln Leu Lys Asp Ser Ile Asp Arg Glu     210 215 220 Lys Ile Ala Val Ile Gly His Ser Phe Gly Gly Ala Thr Val Ile Gln 225 230 235 240 Thr Leu Ser Glu Asp Gln Arg Phe Arg Cys Gly Ile Ala Leu Asp Ala                 245 250 255 Trp Met Phe Pro Leu Gly Asp Glu Val Tyr Ser Arg Ile Pro Gln Pro             260 265 270 Leu Phe Phe Ile Asn Ser Glu Tyr Phe Gln Tyr Pro Ala Asn Ile Ile         275 280 285 Lys Met Lys Lys Cys Tyr Ser Pro Asp Lys Glu Arg Lys Met Ile Thr     290 295 300 Ile Arg Gly Ser Val His Gln Asn Phe Ala Asp Phe Thr Phe Ala Thr 305 310 315 320 Gly Lys Ile Ile Gly His Met Leu Lys Leu Lys Gly Asp Ile Asp Ser                 325 330 335 Asn Val Ala Ile Asp Leu Ser Asn Lys Ala Ser Leu Ala Phe Leu Gln             340 345 350 Lys His Leu Gly Leu His Lys Asp Phe Asp Gln Trp Asp Cys Leu Ile         355 360 365 Glu Gly Asp Asp Glu Asn Leu Ile Pro Gly Thr Asn Ile Asn Thr Thr     370 375 380 Asn Gln His Ile Met Leu Gln Asn Ser Ser Gly Ile Glu Lys Tyr Asn 385 390 395 400 Pro Gly Asn Glu Ser Glu Pro Asn Asn Asp Phe Glu Lys Ala Asn Gln                 405 410 415 Ile Ala Lys Ser Asn Met Leu Val Lys Gly Thr Leu Ser Glu Glu Asp             420 425 430 Tyr Ser Asp Lys Tyr Tyr Phe Asp Val Ala Lys Lys Gly Asn Val Lys         435 440 445 Ile Thr Leu Asn Asn Leu Asn Ser Val Gly Ile Thr Trp Thr Leu Tyr     450 455 460 Lys Glu Gly Asp Leu Asn Asn Tyr Val Leu Tyr Ala Thr Gly Asn Asp 465 470 475 480 Gly Thr Glu Leu Lys Gly Glu Lys Thr Leu Glu Pro Gly Arg Tyr Tyr                 485 490 495 Leu Ser Val Tyr Thr Tyr Asp Asn Gln Ser Gly Ala Tyr Thr Val Asn             500 505 510 Val Lys Gly Asn Leu Lys Asn Glu Val Lys Glu Thr Glu Lys Asp Ala         515 520 525 Ile Lys Glu Val Glu Asn Asn Asn Asp Phe Asp Lys Ala Met Lys Val     530 535 540 Asp Ser Asn Ser Lys Ile Val Gly Thr Leu Ser Asn Asp Asp Leu Lys 545 550 555 560 Asp Ile Tyr Ser Ile Asp Ile Gln Asn Pro Ser Asp Leu Asn Ile Val                 565 570 575 Val Glu Asn Leu Asp Asn Ile Lys Met Asn Trp Leu Leu Tyr Ser Ala             580 585 590 Asp Asp Leu Ser Asn Tyr Val Asp Tyr Ala Asn Ala Asp Gly Asn Lys         595 600 605 Leu Ser Asn Thr Cys Lys Leu Asn Pro Gly Lys Tyr Tyr Leu Cys Val     610 615 620 Tyr Gln Phe Glu Asn Ser Gly Thr Gly Asn Tyr Thr Val Asn Leu Gln 625 630 635 640 Asn Lys <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (5 'primer) <400> 21 cccgggccaa taggcactga aaaa 24 <210> 22 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (3 'primer) <400> 22 ctcgagttat cttcctactg aacc 24 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (5 'primer) <400> 23 cccgggacaa caacacctat aact 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (3 'primer) <400> 24 ctcgagttat ttatttaccc ttaa 24 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (5 'primer) <400> 25 cccgggaatg aaagcgagcc aaacaat 27 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (3 'primer) <400> 26 ctcgagttat ttgttttgta agtttac 27 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (5 'primer) <400> 27 atacaagtac tgatggctgc tgca 24 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (3 'primer) <400> 28 attgtatttc tctattcctg aaga 24 <210> 29 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: tandem myc-tag       sequence <400> 29 gatctgaaca aaaactcatc tcagaagagg atctgaatgg atctgaacaa aaactcatct 60 cagaagagga tctgaatgga tc 82 <210> 30 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (5 'primer) <400> 30 cgtcctctcg agcgatacaa gtactgatgg ctgctgca 38 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR       (3 'primer) <400> 31 cgaccgctcg agctaattgt atttctctat tcctgaaga 39

[Brief description of drawings]

FIG. 1 gCBPAF-AH (lanes 5-8) and control (pGEX
-NM2-PAF-AH introduced E. coli BL21) (lane 1
The figure which shows SDS-PAGE of the sample in each purification step of 4).

FIG. 2 shows samples of gCB (NM2) PAF-AH at each purification step.
The figure which shows SDS-PAGE.

FIG. 3 is a diagram showing the collagen binding ability of GST-gCBPAF-AH and GST-gCB (NM2) PAF-AH.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/18 ZNA C12N 5/00 AF term (reference) 4B024 AA01 BA11 BA80 CA04 CA07 DA02 DA05 DA11 EA04 HA08 4B050 CC05 DD11 LL01 4B065 AA01X AA57X AA87X AA93Y AB01 CA31 CA44

Claims (9)

[Reference number] J200103200 [Claims] 1. A physiologically active protein having a property of binding to collagen and having a platelet activating factor acetylhydrolase activity. 2. A collagen binding region having the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 8 or substitution, deletion or insertion of one or more amino acids therewith. , Or a physiologically active protein in which a region consisting of a transposed amino acid sequence is bound. 3. The physiologically active protein according to claim 2, which has a platelet activating factor acetylhydrolase activity. 4. A nucleic acid encoding the physiologically active protein according to claim 1. 5. A DNA encoding the physiologically active protein according to any one of claims 1 to 3. 6. A vector having the nucleic acid according to claim 5. 7. A DNA consisting of the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13 or a DNA which hybridizes to a nucleotide sequence complementary thereto under stringent conditions, and a nucleotide of SEQ ID NO: 7. A vector which has, in the same readout region, a DNA consisting of a sequence or a DNA hybridizing to a DNA consisting of a base sequence complementary thereto under stringent conditions. 8. A recombinant transformed with the vector according to claim 6 or 7. 9. A method for producing a physiologically active protein, which comprises growing the recombinant according to claim 8 and obtaining a physiologically active protein from the grown product.