Catalase enzymes derived from bacteria from the genera Alcaligenes (Deleya) and Microscilla are disclosed. The enzymes are produced from native or recombinant host cells and can be utilized to destroy or detect hydrogen peroxide, e.g., in production of glyoxylic acid and in glucose sensors, and in processes where hydrogen peroxide is used as a bleaching or antibacterial agent, e.g. in contact lens cleaning, in bleaching steps in pulp and paper preparation and in the pasteurization of dairy products.

This invention relates to newly identified polynucleotides, polypeptides 
encoded by such polynucleotides, the use of such polynucleotides and 
polypeptides, as well as the production and isolation of such 
polynucleotides and polypeptides. More particularly, the polynucleotides 
and polypeptides of the present invention have been putatively identified 
as catalases. 
Generally, in processes where hydrogen peroxide is a by-product, catalases 
can be used to destroy or detect hydrogen peroxide, e.g., in production of 
glyoxylic acid and in glucose sensors. Also, in processes where hydrogen 
peroxide is used as a bleaching or antibacterial agent, catalases can be 
used to destroy residual hydrogen peroxide, e.g. in contact lens cleaning, 
in bleaching steps in pulp and paper preparation and in the pasteurization 
of dairy products. Further, such catalases can be used as catalysts for 
oxidation reactions, e.g., epoxidation and hydroxylation. 
In accordance with one aspect of the present invention, there are provided 
novel enzymes, as well as active fragments, analogs and derivatives 
thereof. 
In accordance with another aspect of the present invention, there are 
provided isolated nucleic acid molecules encoding the enzymes of the 
present invention including mRNAs, cDNAs, genomic DNAs as well as active 
analogs and fragments of such enzymes. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for producing such polypeptides by recombinant 
techniques comprising culturing recombinant prokaryotic and/or eukaryotic 
host cells, containing a nucleic acid sequence of the present invention, 
under conditions promoting expression of said enzymes and subsequent 
recovery of said enzymes 
In accordance with yet a further aspect of the present invention, there are 
also provided nucleic acid probes comprising nucleic acid molecules of 
sufficient length to specifically hybridize to a nucleic acid sequence of 
the present invention. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such enzymes, or polynucleotides encoding 
such enzymes, for in vitro purposes related to scientific research, for 
example, to generate probes for identifying similar sequences which might 
encode similar enzymes from other organisms by using certain regions, 
i.e., conserved sequence regions, of the nucleotide sequence. 
In accordance with yet a further aspect of the present invention, there is 
provided antibodies to such catalases. These antibodies are as probes to 
screen libraries from these or other organisms for members of the 
libraries which could have the same catalase activity or a cross reactive 
activity. 
These and other aspects of the present invention should be apparent to 
those skilled in the art from the teachings herein.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
In order to facilitate understanding of the following description and 
examples which follow certain frequently occurring methods and/or terms 
will be described. 
The term "isolated" means altered "by the hand of man" from its natural 
state; i.e., if it occurs in nature, it has been changed or removed from 
its original environment, or both. For example, a naturally occurring 
polynucleotide or a polypeptide naturally present in a living animal in 
its natural state is not "isolated", but the same polynucleotide or 
polypeptide separated from the coexisting materials of its natural state 
is "isolated", as the term is employed herein. For example, with respect 
to polynucleotides, the term isolated means that it is separated from the 
nucleic acid and cell in which it naturally occurs. 
As part of or following isolation, such polynucleotides can be joined to 
other polynucleotides, such as DNAs, for mutagenesis, to form fusion 
proteins, and for propagation or expression in a host, for instance. The 
isolated polynucleotides, alone or joined to other polynucleotides such as 
vectors, can be introduced into host cells, in culture or in whole 
organisms. Introduced into host cells in culture or in whole organisms, 
such polynucleotides still would be isolated, as the term is used herein, 
because they would not be in their naturally occurring form or 
environment. Similarly, the polynucleotides and polypeptides may occur in 
a composition, such as a media formulation (solutions for introduction of 
polynucleotides or polypeptides, for example, into cells or compositions 
or solutions for chemical or enzymatic reactions which are not naturally 
occurring compositions) and, therein remain isolated polynucleotides or 
polypeptides within the meaning of that term as it is employed herein. 
The term "ligation" refers to the process of forming phosphodiester bonds 
between two or more polynucleotides, which most often are double stranded 
DNAs. Techniques for ligation are well known to the art and protocols for 
ligation are described in standard laboratory manuals and references, such 
as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 
(1989). 
The term "gene" means the segment of DNA involved in 4producing a 
polypeptide chain; it includes regions preceding and following the coding 
region (leader and trailer) as well as intervening sequences (introns) 
between individual coding segments (exons). 
A coding sequence is "operably linked to" another coding sequence when RNA 
polymerase will transcribe the two coding sequences into a single mRNA, 
which is then translated into a single polypeptide having amino acids 
derived from both coding sequences. The coding sequences need not be 
contiguous to one another so long as the expressed sequences ultimately 
process to produce the desired protein. 
"Recombinant" enzymes refer to enzymes produced by recombinant DNA 
techniques; i.e., produced from cells transformed by an exogenous DNA 
construct encoding the desired enzyme. "Synthetic" enzymes are those 
prepared by chemical synthesis. 
A DNA "coding sequence of" or a "nucleotide sequence encoding" a particular 
enzyme, is a DNA sequence which is transcribed and translated into an 
enzyme when placed under the control of appropriate regulatory sequences. 
"Plasmids" are designated by a lower case "p" preceded and/or followed by 
capital letters and/or numbers. The starting plasmids herein are either 
commercially available, publicly available on an unrestricted basis, or 
can be constructed from available plasmids in accord with published 
procedures. In addition, equivalent plasmids to those described are known 
in the art and will be apparent to the ordinarily skilled artisan. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with a 
restriction enzyme that acts only at certain sequences in the DNA. The 
various restriction enzymes used herein are commercially available and 
their reaction conditions, cofactors and other requirements were used as 
would be known to the ordinarily skilled artisan. For analytical purposes, 
typically 1 .mu.g of plasmid or DNA fragment is used with about 2 units of 
enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating 
DNA fragments for plasmid construction, typically 5 to 50 .mu.g of DNA are 
digested with 20 to 250 units of enzyme in a larger volume. Appropriate 
buffers and substrate amounts for particular restriction enzymes are 
specified by the manufacturer. Incubation times of about 1 hour at 
37.degree. C. are ordinarily used, but may vary in accordance with the 
supplier's instructions. After digestion the reaction is electrophoresed 
directly on a polyacrylamide gel to isolate the desired fragment. 
Size separation of the cleaved fragments is performed using 8 percent 
polyacrylamide gel described by Goeddel et al., Nucleic Acids Res., 8:4057 
(1980). 
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide 
or two complementary polydeoxynucleotide strands which may be chemically 
synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus 
will not ligate to another oligonucleotide without adding a phosphate with 
an ATP in the presence of a kinase. A synthetic oligonucleotide will 
ligate to a fragment that has not been dephosphorylated. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 
146). Unless otherwise provided, ligation may be accomplished using known 
buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. 
Unless otherwise stated, transformation was performed as described in 
Sambrook and Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring 
Harbor Laboratory, 1989. 
In accordance with an aspect of the present invention, there are provided 
isolated nucleic acids (polynucleotides) which encode for the mature 
enzyme having the deduced amino acid sequence of FIG. 1 (SEQ ID NO: 6). 
In accordance with another aspect of the present invention, there are 
provided isolated nucleic acids (polynucleotides) which encode for the 
mature enzyme having the deduced amino acid sequence of FIG. 2 (SEQ ID NO: 
8). 
The polynucleotides of this invention were originally recovered from a 
genomic gene library derived from two sources. The first, Alcaligenes 
(Delaya) aquamarinus, is a .beta.-Proteobacteria. It is a gram-negative 
rod that grows optimally at 26.degree. C. and pH 7.2. The second, 
Microscilla furvescens, is a Cytophagales (Bacteria) isolated from Samoa. 
It is a gram-negative rod with gliding motility that grows optimally at 
30.degree. C. and pH 7.0. 
With respect to Alcaligenes (Delaya) aquamarinus, the protein with the 
closest amino acid sequence identity of which the inventors are currently 
aware is the Microscilla furvescens catalase (59.5% protein identity; 60% 
DNA identity). The next closest is a Mycobacterium tuberculosis catalase 
(KatG), with a 54% protein identity. 
With respect to Microscilla furvescens, the protein with the closest amino 
acid sequence identity of which the inventors are currently aware is 
catalase I of Bacillus stearothermophilis, which has a 69% amino acid 
identity. 
Accordingly, the polynucleotides and enzymes encoded thereby are identified 
by the organism from which they were isolated. Such are sometimes referred 
to below as "64CA2" (FIG. 1 and SEQ ID NOS: 5 and 6) and "53CA1" (FIG. 2 
and SEQ ID NOS: 7 and 8). 
One means for isolating the nucleic acid molecules encoding the enzymes of 
the present invention is to probe a gene library with a natural or 
artificially designed probe using art recognized procedures (see, for 
example: Current Protocols in Molecular Biology, Ausubel F. M. et al. 
(EDS.) Green Publishing Company Assoc. and John Wiley Interscience, New 
York, 1989, 1992). It is appreciated by one skilled in the art that the 
polynucleotides of SEQ ID NOS: 5 and 7, or fragments thereof (comprising 
at least 12 contiguous nucleotides), are particularly useful probes. Other 
particularly useful probes for this purpose are hybridizable fragments of 
the sequences of SEQ ID NOS: 5 and 7 (i.e., comprising at least 12 
contiguous nucleotides). 
With respect to nucleic acid sequences which hybridize to specific nucleic 
acid sequences disclosed herein, hybridization may be carried out under 
conditions of reduced stringency, medium stringency or even stringent 
conditions. As an example of oligonucleotide hybridization, a polymer 
membrane containing immobilized denatured nucleic acids is first 
prehybridized for 30 minutes at 45.degree. C. in a solution consisting of 
0.9 M NaCl, 50 mM NaH.sub.2 PO.sub.4, pH 7.0, 5.0 mM Na.sub.2 EDTA, 0.5% 
SDS, 10.times.Denhardt's, and 0.5 mg/mL polyriboadenylic acid. 
Approximately 2.times.10.sup.7 cpm (specific activity 4-9.times.10.sup.8 
cpm/ug) of .sup.32 P end-labeled oligonucleotide probe are then added to 
the solution. After 12-16 hours of incubation, the membrane is washed for 
30 minutes at room temperature in 1.times.SET (150 mM NaCl, 20 mM Tris 
hydrochloride, pH 7.8, 1 mM Na.sub.2 EDTA) containing 0.5% SDS, followed 
by a 30 minute wash in fresh 1.times.SET at (Tm less 10.degree. C.) for 
the oligonucleotide probe. The membrane is then exposed to 
auto-radiographic film for detection of hybridization signals. 
Stringent conditions means hybridization will occur only if there is at 
least 90% identity, preferably at least 95% identity and most preferably 
at least 97% identity between the sequences. Further, it is understood 
that a section of a 100 bps sequence that is 95 bps in length has 95% 
identity with the 1090 bps sequence from which it is obtained. See J. 
Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold 
Spring Harbor Laboratory (1989) which is hereby incorporated by reference 
in its entirety. Also, it is understood that a fragment of a 100 bps 
sequence that is 95 bps in length has 95% identity with the 100 bps 
sequence from which it is obtained. 
As used herein, a first DNA (RNA) sequence is at least 70% and preferably 
at least 80% identical to another DNA (RNA) sequence if there is at least 
70% and preferably at least a 80% or 90% identity, respectively, between 
the bases of the first sequence and the bases of the another sequence, 
when properly aligned with each other, for example when aligned by BLASTN. 
The present invention relates to polynucleotides which differ from the 
reference polynucleotide such that the differences are silent, for 
example, the amino acid sequence encoded by the polynucleotides is the 
same. The present invention also relates to nucleotide changes which 
result in amino acid substitutions, additions, deletions, fusions and 
truncations in the polypeptide encoded by the reference polynucleotide. In 
a preferred aspect of the invention these polypeptides retain the same 
biological action as the polypeptide encoded by the reference 
polynucleotide. 
The polynucleotides of this invention were recovered from genomic gene 
libraries from the organisms identified above. Gene libraries were 
generated from a Lambda ZAP II cloning vector (Stratagene Cloning 
Systems). Mass excisions were performed on these libraries to generate 
libraries in the pBluescript phagemid. Libraries were generated and 
excisions were performed according to the protocols/methods hereinafter 
described. 
The polynucleotides of the present invention may be in the form of RNA or 
DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may 
be double-stranded or single-stranded, and if single stranded may be the 
coding strand or non-coding (anti-sense) strand. The coding sequences 
which encodes the mature enzymes may be identical to the coding sequences 
shown in FIGS. 1-2 (SEQ ID NOS: 5 & 7) or may be a different coding 
sequence which coding sequence, as a result of the redundancy or 
degeneracy of the genetic code, encodes the same mature enzymes as the DNA 
of FIGS. 1-2 (SEQ ID NOS: 6 & 8). 
The polynucleotide which encodes for the mature enzyme of FIGS. 1-2 (SEQ ID 
NOS: 6 & 8) may include, but is not limited to: only the coding sequence 
for the mature enzyme; the coding sequence for the mature enzyme and 
additional coding sequence such as a leader sequence or a proprotein 
sequence; the coding sequence for the mature enzyme (and optionally 
additional coding sequence) and non-coding sequence, such as introns or 
non-coding sequence 5' and/or 3' of the coding sequence for the mature 
enzyme. 
Thus, the term "polynucleotide encoding an enzyme (protein)" encompasses a 
polynucleotide which includes only coding sequence for the enzyme as well 
as a polynucleotide which includes additional coding and/or non-coding 
sequence. 
The present invention further relates to variants of the hereinabove 
described polynucleotides which encode for fragments, analogs and 
derivatives of the enzymes having the deduced amino acid sequences of 
FIGS. 1-2 (SEQ ID NOS: 6 & 8). The variant of the polynucleotide may be a 
naturally occurring allelic variant of the polynucleotide or a 
non-naturally occurring variant of the polynucleotide. 
Thus, the present invention includes polynucleotides encoding the same 
mature enzymes as shown in FIGS. 1-2 (SEQ ID NOS: 6 & 8) as well as 
variants of such polynucleotides which variants encode for a fragment, 
derivative or analog of the enzymes of FIGS. 1-2 (SEQ ID NOS: 6 & 8). Such 
nucleotide variants include deletion variants, substitution variants and 
addition or insertion variants. 
As hereinabove indicated, the polynucleotides may have a coding sequence 
which is a naturally occurring allelic variant of the coding sequences 
shown in FIGS. 1-2 (SEQ ID NOS: 5 & 7). As known in the art, an allelic 
variant is an alternate form of a polynucleotide sequence which may have a 
substitution, deletion or addition of one or more nucleotides, which does 
not substantially alter the function of the encoded enzyme. Also, using 
directed and other evolution strategies, one may make very minor changes 
in DNA sequence which can result in major changes in function. 
Fragments of the full length gene of the present invention may be used as 
hybridization probes for a cDNA or a genomic library to isolate the full 
length DNA and to isolate other DNAs which have a high sequence similarity 
to the gene or similar biological activity. Probes of this type preferably 
have at least 10, preferably at least 15, and even more preferably at 
least 30 bases and may contain, for example, at least 50 or more bases. In 
fact, probes of this type having at least up to 150 bases or greater may 
be preferably utilized. The probe may also be used to identify a DNA clone 
corresponding to a full length transcript and a genomic clone or clones 
that contain the complete gene including regulatory and promotor regions, 
exons and introns. An example of a screen comprises isolating the coding 
region of the gene by using the known DNA sequence to synthesize an 
oligonucleotide probe. Labeled oligonucleotides having a sequence 
complementary or identical to that of the gene or portion of the gene 
sequences of the present invention are used to screen a library of genomic 
DNA to determine which members of the library the probe hybridizes to. 
It is also appreciated that such probes can be and are preferably labeled 
with an analytically detectable reagent to facilitate identification of 
the probe. Useful reagents include but are not limited to radioactivity, 
fluorescent dyes or enzymes capable of catalyzing the formation of a 
detectable product. The probes are thus useful to isolate complementary 
copies of DNA from other sources or to screen such sources for related 
sequences. 
The present invention further relates to polynucleotides which hybridize to 
the hereinabove-described sequences if there is at least 70%, preferably 
at least 90%, and more preferably at least 95% identity between the 
sequences. (As indicated above, 70% identity would include within such 
definition a 70 bps fragment taken from a 100 bp polynucleotide, for 
example.) The present invention particularly relates to polynucleotides 
which hybridize under stringent conditions to the hereinabove-described 
polynucleotides. As herein used, the term "stringent conditions" means 
hybridization will occur only if there is at least 95% and preferably at 
least 97% identity between the sequences. The polynucleotides which 
hybridize to the hereinabove described polynucleotides in a preferred 
embodiment encode enzymes which either retain substantially the same 
biological function or activity as the mature enzyme encoded by the DNA of 
FIGS. 1-2 (SEQ ID NOS: 5 & 7). In referring to identity in the case of 
hybridization, as known in the art, such identity refers to the 
complementarity of two polynucleotide segments. 
Alternatively, the polynucleotide may have at least 15 bases, preferably at 
least 30 bases, and more preferably at least 50 bases which hybridize to 
any part of a polynucleotide of the present invention and which has an 
identity thereto, as hereinabove described, and which may or may not 
retain activity. For example, such polynucleotides may be employed as 
probes for the polynucleotides of SEQ ID NOS: 5 & 7, for example, for 
recovery of the polynucleotide or as a diagnostic probe or as a PCR 
primer. 
Thus, the present invention is directed to polynucleotides having at least 
a 70% identity, preferably at least 90% identity and more preferably at 
least a 95% identity to a polynucleotide which encodes the enzymes of SEQ 
ID NOS: 6 & 8 as well as fragments thereof, which fragments have at least 
15 bases, preferably at least 30 bases, more preferably at least 50 bases 
and most preferably fragments having up to at least 150 bases or greater, 
which fragments are at least 90% identical, preferably at least 95% 
identical and most preferably at least 97% identical to any portion of a 
polynucleotide of the present invention. 
The terms "fragment," "derivative" and "analog" when referring to the 
enzymes of FIGS. 1-2 (SEQ ID NOS. 6& 8) means enzymes which retain 
essentially the same biological function or activity as such enzymes. 
Thus, an analog includes a proprotein which can be activated by cleavage 
of the proprotein portion to produce an active mature enzyme. 
The enzymes of the present invention may be a recombinant enzyme, a natural 
enzyme or a synthetic enzyme, preferably a recombinant enzyme. 
The fragment, derivative or analog of the enzymes of FIGS. 1-2 (SEQ ID NOS: 
6 & 8) may be (i) one in which one or more of the amino acid residues are 
substituted with a conserved or non-conserved amino acid residue 
(preferably a conserved amino acid residue) and such substituted amino 
acid residue may or may not be one encoded by the genetic code, or (ii) 
one in which one or more of the amino acid residues includes a substituent 
group, or (iii) one in which the mature enzyme is fused with another 
compound, such as a compound to increase the half-life of the enzyme (for 
example, polyethylene glycol), or (iv) one in which the additional amino 
acids are fused to the mature enzyme, such as a leader or secretory 
sequence or a sequence which is employed for purification of the mature 
enzyme or a proprotein sequence. Such fragments, derivatives and analogs 
are deemed to be within the scope of those skilled in the art from the 
teachings herein. 
The enzymes and polynucleotides of the present invention are preferably 
provided in an isolated form, and preferably are purified to homogeneity. 
The present invention also relates to vectors which include polynucleotides 
of the present invention, host cells which are genetically engineered with 
vectors of the invention and the production of enzymes of the invention by 
recombinant techniques. 
Host cells are genetically engineered (transduced or transformed or 
transfected) with the vectors of this invention which may be, for example, 
a cloning vector such as an expression vector. The vector may be, for 
example, in the form of a plasmid, a phage, etc. The engineered host cells 
can be cultured in conventional nutrient media modified as appropriate for 
activating promoters, selecting transformants or amplifying the genes of 
the present invention. The culture conditions, such as temperature, pH and 
the like, are those previously used with the host cell selected for 
expression, and will be apparent to the ordinarily skilled artisan. 
The polynucleotides of the present invention may be employed for producing 
enzymes by recombinant techniques. Thus, for example, the polynucleotide 
may be included in any one of a variety of expression vectors for 
expressing an enzyme. Such vectors include chromosomal, nonchromosomal and 
synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; 
phage DNA; baculovirus; yeast plasmids; vectors derived from combinations 
of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl 
pox virus, and pseudorabies. However, any other vector may be used as long 
as it is replicable and viable in the host. 
The appropriate DNA sequence may be inserted into the vector by a variety 
of procedures. In general, the DNA sequence is inserted into an 
appropriate restriction endonuclease site(s) by procedures known in the 
art. Such procedures and others are deemed to be within the scope of those 
skilled in the art. 
The DNA sequence in the expression vector is operatively linked to an 
appropriate expression control sequence(s) (promoter) to direct mRNA 
synthesis. As representative examples of such promoters, there may be 
mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda 
P.sub.L promoter and other promoters known to control expression of genes 
in prokaryotic or eukaryotic cells or their viruses. The expression vector 
also contains a ribosome binding site for translation initiation and a 
transcription terminator. The vector may also include appropriate 
sequences for amplifying expression. 
In addition, the expression vectors preferably contain one or more 
selectable marker genes to provide a phenotypic trait for selection of 
transformed host cells such as dihydrofolate reductase or neomycin 
resistance for eukaryotic cell culture, or such as tetracycline or 
ampicillin resistance in E. coli. 
The vector containing the appropriate DNA sequence as hereinabove 
described, as well as an appropriate promoter or control sequence, may be 
employed to transform an appropriate host to permit the host to express 
the protein. 
As representative examples of appropriate hosts, there may be mentioned: 
bacterial cells, such as E. coli, Streptomyces, Bacillus subtilis; fungal 
cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera 
Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant 
cells, etc. The selection of an appropriate host is deemed to be within 
the scope of those skilled in the art from the teachings herein. 
More particularly, the present invention also includes recombinant 
constructs comprising one or more of the sequences as broadly described 
above. The constructs comprise a vector, such as a plasmid or viral 
vector, into which a sequence of the invention has been inserted, in a 
forward or reverse orientation. In a preferred aspect of this embodiment, 
the construct further comprises regulatory sequences, including, for 
example, a promoter, operably linked to the sequence. Large numbers of 
suitable vectors and promoters are known to those of skill in the art, and 
are commercially available. The following vectors are provided by way of 
example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBluescript II 
KS(Stratagene), ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: 
pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL SV40 (Pharmacia). However, 
any other plasmid or vector may be used as long as they are replicable and 
viable in the host. 
Promoter regions can be selected from any desired gene using CAT 
(chloramphenicol transferase) vectors or other vectors with selectable 
markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named 
bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, 
P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSV 
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse 
metallothionein-I. Selection of the appropriate vector and promoter is 
well within the level of ordinary skill in the art. 
In a further embodiment, the present invention relates to host cells 
containing the above-described constructs. The host cell can be a higher 
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, 
such as a yeast cell, or the host cell can be a prokaryotic cell, such as 
a bacterial cell. Introduction of the construct into the host cell can be 
effected by calcium phosphate transfection, DEAE-Dextran mediated 
transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic 
Methods in Molecular Biology, (1986)). 
The constructs in host cells can be used in a conventional manner to 
produce the gene product encoded by the recombinant sequence. 
Alternatively, the enzymes of the invention can be synthetically produced 
by conventional peptide synthesizers. 
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the present invention. Appropriate 
cloning and expression vectors for use with prokaryotic and eukaryotic 
hosts are described by Sambrook et al., Molecular Cloning: A Laboratory 
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure 
of which is hereby incorporated by reference. 
Transcription of the DNA encoding the enzymes of the present invention by 
higher eukaryotes is increased by inserting an enhancer sequence into the 
vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 
300 bp that act on a promoter to increase its transcription. Examples 
include the SV40 enhancer on the late side of the replication origin bp 
100 to 270, a cytomegalovirus early promoter enhancer, the polyoma 
enhancer on the late side of the replication origin, and adenovirus 
enhancers. 
Generally, recombinant expression vectors will include origins of 
replication and selectable markers permitting transformation of the host 
cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae 
TRP1 gene, and a promoter derived from a highly-expressed gene to direct 
transcription of a downstream structural sequence. Such promoters can be 
derived from operons encoding glycolytic enzymes such as 
3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, or heat 
shock proteins, among others. The heterologous structural sequence is 
assembled in appropriate phase with translation initiation and termination 
sequences, and preferably, a leader sequence capable of directing 
secretion of translated enzyme. Optionally, the heterologous sequence can 
encode a fusion enzyme including an N-terminal identification peptide 
imparting desired characteristics, e.g., stabilization or simplified 
purification of expressed recombinant product. 
Useful expression vectors for bacterial use are constructed by inserting a 
structural DNA sequence encoding a desired protein together with suitable 
translation initiation and termination signals in operable reading phase 
with a functional promoter. The vector will comprise one or more 
phenotypic selectable markers and an origin of replication to ensure 
maintenance of the vector and to, if desirable, provide amplification 
within the host. Suitable prokaryotic hosts for transformation include E. 
coli, Bacillus subtilis, Salmonella typhimurium and various species within 
the genera Pseudomonas, Streptomyces, and Staphylococcus, although others 
may also be employed as a matter of choice. 
As a representative but nonlimiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and pGEM 1 (Promega Biotec, Madison, Wis., 
USA). These pBR322 "backbone" sections are combined with an appropriate 
promoter and the structural sequence to be expressed. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, the selected promoter is induced by 
appropriate means (e.g., temperature shift or chemical induction) and 
cells are cultured for an additional period. 
Cells are typically harvested by centrifugation, disrupted by physical or 
chemical means, and the resulting crude extract retained for further 
purification. 
Microbial cells employed in expression of proteins can be disrupted by any 
convenient method, including freeze-thaw cycling, sonication, mechanical 
disruption, or use of cell lysing agents, such methods are well known to 
those skilled in the art. 
Various mammalian cell culture systems can also be employed to express 
recombinant protein. Examples of mammalian expression systems include the 
COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 
23:175 (1981), and other cell lines capable of expressing a compatible 
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. 
Mammalian expression vectors will comprise an origin of replication, a 
suitable promoter and enhancer, and also any necessary ribosome binding 
sites, polyadenylation site, splice donor and acceptor sites, 
transcriptional termination sequences, and 5' flanking nontranscribed 
sequences. DNA sequences derived from the SV40 splice, and polyadenylation 
sites may be used to provide the required nontranscribed genetic elements. 
The enzyme can be recovered and purified from recombinant cell cultures by 
methods including ammonium sulfate or ethanol precipitation, acid 
extraction, anion or cation exchange chromatography, phosphocellulose 
chromatography, hydrophobic interaction chromatography, affinity 
chromatography, hydroxylapatite chromatography and lectin chromatography. 
Protein refolding steps can be used, as necessary, in completing 
configuration of the mature protein. Finally, high performance liquid 
chromatography (HPLC) can be employed for final purification steps. 
The enzymes of the present invention may be a naturally purified product, 
or a product of chemical synthetic procedures, or produced by recombinant 
techniques from a prokaryotic or eukaryotic host (for example, by 
bacterial, yeast, higher plant, insect and mammalian cells in culture). 
Depending upon the host employed in a recombinant production procedure, 
the enzymes of the present invention may be glycosylated or may be 
non-glycosylated. Enzymes of the invention may or may not also include an 
initial methionine amino acid residue. 
Antibodies generated against the enzymes corresponding to a sequence of the 
present invention can be obtained by direct injection of the enzymes into 
an animal or by administering the enzymes to an animal, preferably a 
nonhuman. The antibody so obtained will then bind the enzymes itself. In 
this manner, even a sequence encoding only a fragment of the enzymes can 
be used to generate antibodies binding the whole native enzymes. Such 
antibodies can then be used to isolate the enzyme from cells expressing 
that enzyme. 
For preparation of monoclonal antibodies, any technique which provides 
antibodies produced by continuous cell line cultures can be used. Examples 
include the hybridoma technique (Kohler and Milstein, Nature, 256:495-497, 
1975), the trioma technique, the human B-cell hybridoma technique (Kozbor 
et al., Immunology Today 4:72, 1983), and the EBV-hybridoma technique to 
produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies 
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). 
Techniques described for the production of single chain antibodies (U.S. 
Pat. No. 4,946,778) can be adapted to produce single chain antibodies to 
immunogenic enzyme products of this invention. Also, transgenic mice may 
be used to express humanized antibodies to immunogenic enzyme products of 
this invention. 
Antibodies generated against an enzyme of the present invention may be used 
in screening for similar enzymes from other organisms and samples. Such 
screening techniques are known in the art, for example, one such screening 
assay is described in Sambrook and Maniatis, Molecular Cloning: A 
Laboratory Manual (2d Ed.), vol. 2:Section 8.49, Cold Spring Harbor 
Laboratory, 1989, which is hereby incorporated by reference in its 
entirety. 
The present invention will be further described with reference to the 
following examples; however, it is to be understood that the present 
invention is not limited to such examples. All parts or amounts, unless 
otherwise specified, are by weight. 
EXAMPLE 1 
Production of the Expression Gene Bank 
An E. coli catalase negative host strain CAT500 was infected with a phage 
solution containing sheared pieces of DNA from Alcaligenes (Deleya) 
aquamarinus in pBluescript plasmid and plated on agar containing LB with 
ampicillin (100 .mu.g/mL), methicillin (80 .mu.g/mL) and kanamycin (100 
.mu.g/mL) according to the method of Hay and Short (Hay, B. and Short, J., 
J. Strategies, 5:16, 1992). The resulting colonies were picked with 
sterile toothpicks and used to singly inoculate each of the wells of 
96-well microtiter plates. The wells contained 250 .mu.L of SOB media with 
100 .mu.g/mL ampicillin, 80 .mu.g/mL methicillin, and (SOB Amp/Meth/Kan). 
The cells were grown overnight at 37.degree. C. without shaking. This 
constituted generation of the "SourceGeneBank"; each well of the Source 
GeneBank thus contained a stock culture of E. coli cells, each of which 
contained a pBluescript plasmid with a unique DNA insert. Same protocol 
was adapted for screening catalase from Microscilla furvescens. 
EXAMPLE 2 
Screening for Catalase Activity 
The plates of the Source GeneBank were used to multiply inoculate a single 
plate (the "Condensed Plate") containing in each well 200 .mu.L of SOB 
Amp/Meth/Kan. This step was performed using the High Density Replicating 
Tool (HDRT) of the Beckman Biomek with a 1% bleach, water, isopropanol, 
air-dry sterilization cycle in between each inoculation. Each well of the 
Condensed Plate thus contained 4 different pBluescript clones from each of 
the source library plates. Nine such condensed plates were prepared and 
grown for 16 h at 37.degree. C. 
One hundred (100) .mu.L of the overnight culture was transferred to the 
white polyfiltronic assay plates containing 100 .mu.L Hepes/well. A 0.03% 
solution of hydrogen peroxide was made in 5% Triton and 20 .mu.L of this 
solution was added to each well. The plates were incubated at room 
temperature for one hour. After an hour, 50 .mu.L of 120 mM 
3-(p-hydroxyphenyl)-propionic acid and 1 unit of horseradish peroxidase 
were added to each well and the plates were incubated at room temperature 
for 1 hour. To quench the reaction, 50 .mu.L of 1 M Tris-base was added to 
each well. The wells were excited on a fluorometer at 320 nm and read at 
404 nm. A low value signified a positive catalase hit. 
EXAMPLE 3 
Isolation and Purification of the Active Clone 
In order to isolate the individual clone which carried the activity, the 
Source GeneBank plates were thawed and the individual wells used to singly 
inoculate a new plate containing SOB Amp/Meth/Kan. As above the plate was 
incubated at 37.degree. C. to grow the cells, and assayed for activity as 
described above. Once the active well from the source plate was 
identified, the cells from the source plate were streaked on agar with 
LB/Amp/Meth/Kan and grown overnight at 37.degree. C. to obtain single 
colonies. Eight single colonies were picked with a sterile toothpick and 
used to singly inoculate the wells of a 96-well microtiter plate. The 
wells contained 250 .mu.L of SOB Amp/Meth/Kan. The cells were grown 
overnight at 37.degree. C. without shaking. A 100 .mu.L aliquot was 
removed from each well and assayed as indicated above. The most active 
clone was identified and the remaining 150 .mu.L of culture was used to 
streak an agar plate with LB/Amp/Meth/Kan. Eight single colonies were 
picked, grown and assayed as above. The most active clone was used to 
inoculate 3mL cultures of LB/Amp/Meth/Kan, which were grown overnight. The 
plasmid DNA was isolated from the cultures and utilized for sequencing. 
EXAMPLE 4 
Expression of Catalases 
DNA encoding the enzymes of the present invention, SEQ ID NOS: 6 and 8, 
were initially amplified from a pBluescript vector containing the DNA by 
the PCR technique using the primers noted herein. The amplified sequences 
were then inserted into the respective pQE vector listed beneath the 
primer sequences, and the enzyme was expressed according to the protocols 
set forth herein. The 5' and 3' oligonucleotide primer sequences used for 
subcloning and vectors for the respective genes are as follows: 
__________________________________________________________________________ 
Alcaligenes (Deleya) aquamarinus catalse: (pQET vector) 
5' Primer 
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGAATAACGCATCCGCTGAC EcoRI 
SEQ ID NO: 1 
3' Primer 
CGGAAAGCTTTTACGACGCGACGTCGAAACG HindIII 
SEQ ID NO: 2 
__________________________________________________________________________ 
Microscilla furvescens catalase: (pQET vector) 
5' Primer 
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGAAAATCACAAACACTCA EcoRI 
SEQ ID NO: 3 
3' Primer 
CGAAGGTACCTTATTTCAGATCAAACCGGTC KpnI 
SEQ ID NO: 4 
__________________________________________________________________________ 
The restriction enzyme sites indicated correspond to the restriction enzyme 
sites on the bacterial expression vector indicated for the respective gene 
(Qiagen, Inc. Chatsworth, Calif.). The pQET vector encodes antibiotic 
resistance (Amp.sup.r), a bacterial origin of replication (ori), an 
IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), a 
6-His tag and restriction enzyme sites. 
The pQET vector was digested with the restriction enzymes indicated. The 
amplified sequences were ligated into the respective pQET vector and 
inserted in frame with the sequence encoding for the RBS. The native stop 
codon was incorporated so the genes were not fused to the His tag of the 
vector. The ligation mixture was then used to transform the E. coli strain 
UM255/pREP4 (Qiagen, Inc.) by electroporation. UM255/pREP4 contains 
multiple copies of the plasmid pREP4, which expresses the lad repressor 
and also confers kanamycin resistance (Kan.sup.r). Transformants were 
identified by their ability to grow on LB plates and ampicillin/kanamycin 
resistant colonies were selected. Plasmid DNA was isolated and confirmed 
by restriction analysis. Clones containing the desired constructs were 
grown overnight (O/N) in liquid culture in LB media supplemented with both 
Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture was used to inoculate 
a large culture at a ratio of 1:100 to 1:250. The cells were grown to an 
optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG 
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a final 
concentration of 1 mM. IPTG induces by inactivating the lad repressor, 
clearing the P/O leading to increased gene expression. Cells were grown an 
extra 3 to 4 hours. Cells were then harvested by centrifugation. The 
primer sequences set out above may also be employed to isolate the target 
gene from the deposited material by hybridization techniques described 
above. 
Cited Literature 
1) U.S. Pat. No. 5, 439,813, Aug. 8, 1995, Production of glyoxylic acid 
with glycolate oxidase and catalase immobilized on oxirane acrylic beads, 
Anton, D. L., Wilmington, Del., DiCosimo, R., Wilmington, Del., Gavagan, 
J. E., Wilmington, Del. 
2) U.S. Pat. No.: 5,360,732, Nov.1, 1994, Production of Aspergillus niger 
catalase-R, Berka, R. M., San Mateo, Calif., Fowler, T., Redwood City, 
Calif., Rey, M. W., San Mateo, Calif. 
3) U.S. Pat. No.: 4,460,686, Jul. 17, 1984, Glucose oxidation with 
immobilized glucose oxidase-catalase, Hartmeier, W., Ingelheim am Rhein, 
Germany 
4) U.S. Pat. No.: 5,447,650, Sep. 5, 1995, Composition for preventing the 
accumulation of inorganic deposits on contact lenses, Cafaro, D. P., Santa 
Ana, Calif. 
5) U.S. Pat. No.: 5,362,647, Nov. 8, 1994, Compositions and methods for 
destroying hydrogen peroxide, Cook, J. N., Mission Viejo, Calif., Worsley, 
J. L., Irvine, Calif. 
6) U.S. Pat. No.: 5,266,338, 1993, Cascione, A. S., Rapp, H. 
7) Patrick Dhaese, "Catalase: An Enzyme with Growing Industrial Potential" 
CHIMICA OGGIA/Chemistry Today, January/February, 1996. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 8 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 52 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: pcr primer 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGAATAAC GCATCCGCTG AC - # 
52 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 31 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: pcr primer 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
# 31 CGCG ACGTCGAAAC G 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 52 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: pcr primer 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGGAAAAT CACAAACACT CA - # 
52 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 31 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: pcr primer 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 31 CAGA TCAAACCGGT C 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2262 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...2259 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- ATG AAT AAC GCA TCC GCT GAC GAT CTA CAC AG - #T AGC TTG CAG CAA AGA 
48 
Met Asn Asn Ala Ser Ala Asp Asp Leu His Se - #r Ser Leu Gln Gln Arg 
# 15 
- TGC AGA GCA TTT GTT CCC TTG GTA TCG CCA AG - #G CAT AGA GCA ATA AGG 
96 
Cys Arg Ala Phe Val Pro Leu Val Ser Pro Ar - #g His Arg Ala Ile Arg 
# 30 
- GAG AGA GCT ATG AGC GGT AAA TGT CCT GTC AT - #G CAC GGT GGT AAC ACC 
144 
Glu Arg Ala Met Ser Gly Lys Cys Pro Val Me - #t His Gly Gly Asn Thr 
# 45 
- TCG ACC GGT ACT TCC AAC AAA GAT TGG TGG CC - #G GAA GGG TTG AAC CTG 
192 
Ser Thr Gly Thr Ser Asn Lys Asp Trp Trp Pr - #o Glu Gly Leu Asn Leu 
# 60 
- GAT ATT TTG CAT CAG CAA GAT CGC AAA TCA GA - #C CCG ATG GAT CCG GAT 
240 
Asp Ile Leu His Gln Gln Asp Arg Lys Ser As - #p Pro Met Asp Pro Asp 
# 80 
- TTC AAC TAC CGT GAA GAA GTA CGC AAG CTC GA - #T TTC GAC GCG CTG AAG 
288 
Phe Asn Tyr Arg Glu Glu Val Arg Lys Leu As - #p Phe Asp Ala Leu Lys 
# 95 
- AAA GAT GTC CAC GCG TTG ATG ACC GAT AGC CA - #A GAG TGG TGG CCC GCT 
336 
Lys Asp Val His Ala Leu Met Thr Asp Ser Gl - #n Glu Trp Trp Pro Ala 
# 110 
- GAC TGG GGG CAC TAC GGC GGT TTG ATG ATC CG - #T ATG GCT TGG CAC TCC 
384 
Asp Trp Gly His Tyr Gly Gly Leu Met Ile Ar - #g Met Ala Trp His Ser 
# 125 
- GCT GGC ACC TAC CGT ATT GCT GAT GGC CGT GG - #G GGC GGT GGT ACC GGA 
432 
Ala Gly Thr Tyr Arg Ile Ala Asp Gly Arg Gl - #y Gly Gly Gly Thr Gly 
# 140 
- AGC CAG CGC TTT GCA CCG CTC AAC TCC TGG CC - #G GAC AAC GTC AGC CTG 
480 
Ser Gln Arg Phe Ala Pro Leu Asn Ser Trp Pr - #o Asp Asn Val Ser Leu 
145 1 - #50 1 - #55 1 - 
#60 
- GAT AAA GCG CGC CGT CTG CTG TGG CCG ATC AA - #G AAG AAG TAC GGC AAC 
528 
Asp Lys Ala Arg Arg Leu Leu Trp Pro Ile Ly - #s Lys Lys Tyr Gly Asn 
# 175 
- AAA ATC AGC TGG GCA GAC CTG ATG ATT CTG GC - #T GGC ACC GTG GCT TAT 
576 
Lys Ile Ser Trp Ala Asp Leu Met Ile Leu Al - #a Gly Thr Val Ala Tyr 
# 190 
- GAG TCC ATG GGC TTA CCT GCT TAC GGC TTC TC - #T TTC GGC CGC GTC GAT 
624 
Glu Ser Met Gly Leu Pro Ala Tyr Gly Phe Se - #r Phe Gly Arg Val Asp 
# 205 
- ATT TGG GAA CCC GAA AAA GAT ATC TAC TGG GG - #T GAC GAA AAA GAG TGG 
672 
Ile Trp Glu Pro Glu Lys Asp Ile Tyr Trp Gl - #y Asp Glu Lys Glu Trp 
# 220 
- CTG GCA CCT TCT GAC GAA CGC TAC GGC GAC GT - #G AAC AAG CCA GAG ACC 
720 
Leu Ala Pro Ser Asp Glu Arg Tyr Gly Asp Va - #l Asn Lys Pro Glu Thr 
225 2 - #30 2 - #35 2 - 
#40 
- ATG GAA AAC CCG CTG GCG GCT GTC CAA ATG GG - #T CTG ATC TAT GTG AAC 
768 
Met Glu Asn Pro Leu Ala Ala Val Gln Met Gl - #y Leu Ile Tyr Val Asn 
# 255 
- CCG GAA GGT GTT AAC GGC CAC CCT GAT CCG CT - #G AGA ACC GCA CAG CAG 
816 
Pro Glu Gly Val Asn Gly His Pro Asp Pro Le - #u Arg Thr Ala Gln Gln 
# 270 
- GTA CTT GAA ACC TTC GCC CGT ATG GCG ATG AA - #C GAC GAA AAA ACC GCA 
864 
Val Leu Glu Thr Phe Ala Arg Met Ala Met As - #n Asp Glu Lys Thr Ala 
# 285 
- GCC CTC ACA GCT GGC GGC CAC ACC GTC GGT AA - #T TGT CAC GGT AAT GGC 
912 
Ala Leu Thr Ala Gly Gly His Thr Val Gly As - #n Cys His Gly Asn Gly 
# 300 
- AAT GCC TCT GCG TTA GCC CCT GAC CCA AAA GC - #C TCT GAC GTT GAA AAC 
960 
Asn Ala Ser Ala Leu Ala Pro Asp Pro Lys Al - #a Ser Asp Val Glu Asn 
305 3 - #10 3 - #15 3 - 
#20 
- CAG GGC TTA GGT TGG GGC AAC CCC AAC ATG CA - #G GGC AAG GCA AGC AAC 
1008 
Gln Gly Leu Gly Trp Gly Asn Pro Asn Met Gl - #n Gly Lys Ala Ser Asn 
# 335 
- GCC GTG ACC TCG GGT ATC GAA GGT GCT TGG AC - #C ACC AAC CCC ACG AAA 
1056 
Ala Val Thr Ser Gly Ile Glu Gly Ala Trp Th - #r Thr Asn Pro Thr Lys 
# 350 
- TTC GAT ATG GGC TAT TTC GAC CTG CTG TTC GG - #C TAC AAT TGG GAA CTG 
1104 
Phe Asp Met Gly Tyr Phe Asp Leu Leu Phe Gl - #y Tyr Asn Trp Glu Leu 
# 365 
- AAA AAG AGT CCT GCC GGT GCC CAC CAT TGG GA - #A CCG ATT GAC ATC AAA 
1152 
Lys Lys Ser Pro Ala Gly Ala His His Trp Gl - #u Pro Ile Asp Ile Lys 
# 380 
- AAG GAA AAC AAG CCG GTT GAC GCC AGC GAC CC - #C TCT ATT CGC CAC AAC 
1200 
Lys Glu Asn Lys Pro Val Asp Ala Ser Asp Pr - #o Ser Ile Arg His Asn 
385 3 - #90 3 - #95 4 - 
#00 
- CCG ATC ATG ACC GAT GCG GAT ATG GCG ATA AA - #G GTA AAT CCG ACC TAT 
1248 
Pro Ile Met Thr Asp Ala Asp Met Ala Ile Ly - #s Val Asn Pro Thr Tyr 
# 415 
- CGC GCT ATC TGC GAA AAA TTC ATG GCC GAT CC - #T GAG TAC TTC AAG AAA 
1296 
Arg Ala Ile Cys Glu Lys Phe Met Ala Asp Pr - #o Glu Tyr Phe Lys Lys 
# 430 
- ACT TTC GCG AAG GCG TGG TTC AAG CTG ACG CA - #C CGT GAC CTG GGC CCG 
1344 
Thr Phe Ala Lys Ala Trp Phe Lys Leu Thr Hi - #s Arg Asp Leu Gly Pro 
# 445 
- AAA TCA CGT TAC ATC GGC CCG GAA GTG CCG GC - #A GAA GAC CTG ATT TGG 
1392 
Lys Ser Arg Tyr Ile Gly Pro Glu Val Pro Al - #a Glu Asp Leu Ile Trp 
# 460 
- CAA GAC CCG ATT CCG GCA GGT AAC ACC GAC TA - #C TGC GAA GAA GTG GTC 
1440 
Gln Asp Pro Ile Pro Ala Gly Asn Thr Asp Ty - #r Cys Glu Glu Val Val 
465 4 - #70 4 - #75 4 - 
#80 
- AAG CAG AAA ATT GCA CAA AGT GGC CTG AGC AT - #T AGT GAG ATG GTC TCC 
1488 
Lys Gln Lys Ile Ala Gln Ser Gly Leu Ser Il - #e Ser Glu Met Val Ser 
# 495 
- ACC GCT TGG GAC AGT GCC CGT ACT TAT CGC GG - #T TCC GAT ATG CGC GGC 
1536 
Thr Ala Trp Asp Ser Ala Arg Thr Tyr Arg Gl - #y Ser Asp Met Arg Gly 
# 510 
- GGT GCT AAC GGT GCC CGC ATT CGC TTG GCC CC - #A CAG AAC GAG TGG CAG 
1584 
Gly Ala Asn Gly Ala Arg Ile Arg Leu Ala Pr - #o Gln Asn Glu Trp Gln 
# 525 
- GGC AAC GAG CCG GAG CGC CTG GCG AAA GTG CT - #G AGC GTC TAC GAG CAG 
1632 
Gly Asn Glu Pro Glu Arg Leu Ala Lys Val Le - #u Ser Val Tyr Glu Gln 
# 540 
- ATC TCT GCC GAC ACC GGC GCT AGC ATC GCG GA - #C GTG ATC GTT CTG GCC 
1680 
Ile Ser Ala Asp Thr Gly Ala Ser Ile Ala As - #p Val Ile Val Leu Ala 
545 5 - #50 5 - #55 5 - 
#60 
- GGT AGC GTA GGC ATC GAG AAA GCC GCG AAA GC - #A GCA GGT TAC GAT GTG 
1728 
Gly Ser Val Gly Ile Glu Lys Ala Ala Lys Al - #a Ala Gly Tyr Asp Val 
# 575 
- CGC GTT CCC TTC CTG AAA GGC CGT GGC GAT GC - #G ACC GCC GAG ATG ACC 
1776 
Arg Val Pro Phe Leu Lys Gly Arg Gly Asp Al - #a Thr Ala Glu Met Thr 
# 590 
- GAC GCA GAC TCC TTC GCA CCG CTG GAG CCG CT - #G GCC GAT GGC TTC CGC 
1824 
Asp Ala Asp Ser Phe Ala Pro Leu Glu Pro Le - #u Ala Asp Gly Phe Arg 
# 605 
- AAC TGG CAG AAG AAA GAG TAT GTG GTG AAG CC - #G GAA GAG ATG CTG CTG 
1872 
Asn Trp Gln Lys Lys Glu Tyr Val Val Lys Pr - #o Glu Glu Met Leu Leu 
# 620 
- GAT CGT GCG CAG CTG ATG GGC TTA ACC GGC CC - #G GAA ATG ACC GTG CTG 
1920 
Asp Arg Ala Gln Leu Met Gly Leu Thr Gly Pr - #o Glu Met Thr Val Leu 
625 6 - #30 6 - #35 6 - 
#40 
- CTG GGC GGT ATG CGC GTA CTG GGC ACC AAC TA - #T GGT GGC ACC AAA CAC 
1968 
Leu Gly Gly Met Arg Val Leu Gly Thr Asn Ty - #r Gly Gly Thr Lys His 
# 655 
- GGC GTA TTC ACC GAT TGT GAA GGC CAG TTG AC - #C AAC GAC TTT TTT GTG 
2016 
Gly Val Phe Thr Asp Cys Glu Gly Gln Leu Th - #r Asn Asp Phe Phe Val 
# 670 
- AAC CTG ACC GAT ATG GGG AAC AGC TGG AAG CC - #G GTA GGT AGC AAC GCC 
2064 
Asn Leu Thr Asp Met Gly Asn Ser Trp Lys Pr - #o Val Gly Ser Asn Ala 
# 685 
- TAC GAA ATC CGC GAC CGC AAG ACC GGT GCC GT - #G AAG TGG ACC GCC TCG 
2112 
Tyr Glu Ile Arg Asp Arg Lys Thr Gly Ala Va - #l Lys Trp Thr Ala Ser 
# 700 
- CGG GTG GAT CTG GTA TTT GGT TCC AAC TCG CT - #A CTG CGC TCT TAC GCA 
2160 
Arg Val Asp Leu Val Phe Gly Ser Asn Ser Le - #u Leu Arg Ser Tyr Ala 
705 7 - #10 7 - #15 7 - 
#20 
- GAA GTG TAC GCC CAG GAC GAT AAC GGC GAG AA - #G TTC GTC AGA GAC TTC 
2208 
Glu Val Tyr Ala Gln Asp Asp Asn Gly Glu Ly - #s Phe Val Arg Asp Phe 
# 735 
- GTC GCC GCC TGG ACC AAA GTG ATG AAC GCC GA - #C CGT TTC GAC GTC GCG 
2256 
Val Ala Ala Trp Thr Lys Val Met Asn Ala As - #p Arg Phe Asp Val Ala 
# 750 
# 2262 
Ser 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 753 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (v) FRAGMENT TYPE: internal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- Met Asn Asn Ala Ser Ala Asp Asp Leu His Se - #r Ser Leu Gln Gln Arg 
# 15 
- Cys Arg Ala Phe Val Pro Leu Val Ser Pro Ar - #g His Arg Ala Ile Arg 
# 30 
- Glu Arg Ala Met Ser Gly Lys Cys Pro Val Me - #t His Gly Gly Asn Thr 
# 45 
- Ser Thr Gly Thr Ser Asn Lys Asp Trp Trp Pr - #o Glu Gly Leu Asn Leu 
# 60 
- Asp Ile Leu His Gln Gln Asp Arg Lys Ser As - #p Pro Met Asp Pro Asp 
# 80 
- Phe Asn Tyr Arg Glu Glu Val Arg Lys Leu As - #p Phe Asp Ala Leu Lys 
# 95 
- Lys Asp Val His Ala Leu Met Thr Asp Ser Gl - #n Glu Trp Trp Pro Ala 
# 110 
- Asp Trp Gly His Tyr Gly Gly Leu Met Ile Ar - #g Met Ala Trp His Ser 
# 125 
- Ala Gly Thr Tyr Arg Ile Ala Asp Gly Arg Gl - #y Gly Gly Gly Thr Gly 
# 140 
- Ser Gln Arg Phe Ala Pro Leu Asn Ser Trp Pr - #o Asp Asn Val Ser Leu 
145 1 - #50 1 - #55 1 - 
#60 
- Asp Lys Ala Arg Arg Leu Leu Trp Pro Ile Ly - #s Lys Lys Tyr Gly Asn 
# 175 
- Lys Ile Ser Trp Ala Asp Leu Met Ile Leu Al - #a Gly Thr Val Ala Tyr 
# 190 
- Glu Ser Met Gly Leu Pro Ala Tyr Gly Phe Se - #r Phe Gly Arg Val Asp 
# 205 
- Ile Trp Glu Pro Glu Lys Asp Ile Tyr Trp Gl - #y Asp Glu Lys Glu Trp 
# 220 
- Leu Ala Pro Ser Asp Glu Arg Tyr Gly Asp Va - #l Asn Lys Pro Glu Thr 
225 2 - #30 2 - #35 2 - 
#40 
- Met Glu Asn Pro Leu Ala Ala Val Gln Met Gl - #y Leu Ile Tyr Val Asn 
# 255 
- Pro Glu Gly Val Asn Gly His Pro Asp Pro Le - #u Arg Thr Ala Gln Gln 
# 270 
- Val Leu Glu Thr Phe Ala Arg Met Ala Met As - #n Asp Glu Lys Thr Ala 
# 285 
- Ala Leu Thr Ala Gly Gly His Thr Val Gly As - #n Cys His Gly Asn Gly 
# 300 
- Asn Ala Ser Ala Leu Ala Pro Asp Pro Lys Al - #a Ser Asp Val Glu Asn 
305 3 - #10 3 - #15 3 - 
#20 
- Gln Gly Leu Gly Trp Gly Asn Pro Asn Met Gl - #n Gly Lys Ala Ser Asn 
# 335 
- Ala Val Thr Ser Gly Ile Glu Gly Ala Trp Th - #r Thr Asn Pro Thr Lys 
# 350 
- Phe Asp Met Gly Tyr Phe Asp Leu Leu Phe Gl - #y Tyr Asn Trp Glu Leu 
# 365 
- Lys Lys Ser Pro Ala Gly Ala His His Trp Gl - #u Pro Ile Asp Ile Lys 
# 380 
- Lys Glu Asn Lys Pro Val Asp Ala Ser Asp Pr - #o Ser Ile Arg His Asn 
385 3 - #90 3 - #95 4 - 
#00 
- Pro Ile Met Thr Asp Ala Asp Met Ala Ile Ly - #s Val Asn Pro Thr Tyr 
# 415 
- Arg Ala Ile Cys Glu Lys Phe Met Ala Asp Pr - #o Glu Tyr Phe Lys Lys 
# 430 
- Thr Phe Ala Lys Ala Trp Phe Lys Leu Thr Hi - #s Arg Asp Leu Gly Pro 
# 445 
- Lys Ser Arg Tyr Ile Gly Pro Glu Val Pro Al - #a Glu Asp Leu Ile Trp 
# 460 
- Gln Asp Pro Ile Pro Ala Gly Asn Thr Asp Ty - #r Cys Glu Glu Val Val 
465 4 - #70 4 - #75 4 - 
#80 
- Lys Gln Lys Ile Ala Gln Ser Gly Leu Ser Il - #e Ser Glu Met Val Ser 
# 495 
- Thr Ala Trp Asp Ser Ala Arg Thr Tyr Arg Gl - #y Ser Asp Met Arg Gly 
# 510 
- Gly Ala Asn Gly Ala Arg Ile Arg Leu Ala Pr - #o Gln Asn Glu Trp Gln 
# 525 
- Gly Asn Glu Pro Glu Arg Leu Ala Lys Val Le - #u Ser Val Tyr Glu Gln 
# 540 
- Ile Ser Ala Asp Thr Gly Ala Ser Ile Ala As - #p Val Ile Val Leu Ala 
545 5 - #50 5 - #55 5 - 
#60 
- Gly Ser Val Gly Ile Glu Lys Ala Ala Lys Al - #a Ala Gly Tyr Asp Val 
# 575 
- Arg Val Pro Phe Leu Lys Gly Arg Gly Asp Al - #a Thr Ala Glu Met Thr 
# 590 
- Asp Ala Asp Ser Phe Ala Pro Leu Glu Pro Le - #u Ala Asp Gly Phe Arg 
# 605 
- Asn Trp Gln Lys Lys Glu Tyr Val Val Lys Pr - #o Glu Glu Met Leu Leu 
# 620 
- Asp Arg Ala Gln Leu Met Gly Leu Thr Gly Pr - #o Glu Met Thr Val Leu 
625 6 - #30 6 - #35 6 - 
#40 
- Leu Gly Gly Met Arg Val Leu Gly Thr Asn Ty - #r Gly Gly Thr Lys His 
# 655 
- Gly Val Phe Thr Asp Cys Glu Gly Gln Leu Th - #r Asn Asp Phe Phe Val 
# 670 
- Asn Leu Thr Asp Met Gly Asn Ser Trp Lys Pr - #o Val Gly Ser Asn Ala 
# 685 
- Tyr Glu Ile Arg Asp Arg Lys Thr Gly Ala Va - #l Lys Trp Thr Ala Ser 
# 700 
- Arg Val Asp Leu Val Phe Gly Ser Asn Ser Le - #u Leu Arg Ser Tyr Ala 
705 7 - #10 7 - #15 7 - 
#20 
- Glu Val Tyr Ala Gln Asp Asp Asn Gly Glu Ly - #s Phe Val Arg Asp Phe 
# 735 
- Val Ala Ala Trp Thr Lys Val Met Asn Ala As - #p Arg Phe Asp Val Ala 
# 750 
- Ser 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 2238 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: cDNA 
- (ix) FEATURE: 
(A) NAME/KEY: Coding Se - #quence 
(B) LOCATION: 1...2235 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- ATG GAA AAT CAC AAA CAC TCA GGA TCT TCT AC - #G TAT AAC ACA AAC ACT 
48 
Met Glu Asn His Lys His Ser Gly Ser Ser Th - #r Tyr Asn Thr Asn Thr 
# 15 
- GGC GGA AAA TGC CCT TTT ACC GGA GGT TCG CT - #T AAG CAA AGT GCA GGT 
96 
Gly Gly Lys Cys Pro Phe Thr Gly Gly Ser Le - #u Lys Gln Ser Ala Gly 
# 30 
- GGC GGC ACC AAA AAC AGG GAT TGG TGG CCC AA - #C ATG CTC AAC CTC GGC 
144 
Gly Gly Thr Lys Asn Arg Asp Trp Trp Pro As - #n Met Leu Asn Leu Gly 
# 45 
- ATC TTA CGC CAA CAT TCA TCG CTA TCG GAC CC - #A AAC GAC CCG GAT TTT 
192 
Ile Leu Arg Gln His Ser Ser Leu Ser Asp Pr - #o Asn Asp Pro Asp Phe 
# 60 
- GAC TAT GCC GAA GAG TTT AAG AAG CTA GAT CT - #G GCA GCG GTT AAA AAG 
240 
Asp Tyr Ala Glu Glu Phe Lys Lys Leu Asp Le - #u Ala Ala Val Lys Lys 
# 80 
- GAC CTG GCA GCG CTA ATG ACA GAT TCA CAG GA - #C TGG TGG CCA GCA GAT 
288 
Asp Leu Ala Ala Leu Met Thr Asp Ser Gln As - #p Trp Trp Pro Ala Asp 
# 95 
- TAC GGT CAT TAT GGC CCC TTC TTT ATA CGC AT - #G GCG TGG CAC AGC GCC 
336 
Tyr Gly His Tyr Gly Pro Phe Phe Ile Arg Me - #t Ala Trp His Ser Ala 
# 110 
- GGC ACC TAC CGT ATC GGT GAT GGC CGT GGT GG - #C GGT GGC TCC GGC TCA 
384 
Gly Thr Tyr Arg Ile Gly Asp Gly Arg Gly Gl - #y Gly Gly Ser Gly Ser 
# 125 
- CAG CGC TTC GCG CCT CTC AAT AGC TGG CCA GA - #C AAT GCC AAT CTG GAT 
432 
Gln Arg Phe Ala Pro Leu Asn Ser Trp Pro As - #p Asn Ala Asn Leu Asp 
# 140 
- AAA GCA CGC TTG CTT CTT TGG CCC ATC AAA CA - #A AAA TAC GGT CGA AAA 
480 
Lys Ala Arg Leu Leu Leu Trp Pro Ile Lys Gl - #n Lys Tyr Gly Arg Lys 
145 1 - #50 1 - #55 1 - 
#60 
- ATC TCC TGG GCG GAT CTA ATG ATA CTC ACA GG - #A AAC GTA GCT CTG GAA 
528 
Ile Ser Trp Ala Asp Leu Met Ile Leu Thr Gl - #y Asn Val Ala Leu Glu 
# 175 
- ACT ATG GGC TTT AAA ACT TTT GGT TTT GCA GG - #T GGC AGA GCA GAT GTA 
576 
Thr Met Gly Phe Lys Thr Phe Gly Phe Ala Gl - #y Gly Arg Ala Asp Val 
# 190 
- TGG GAG CCT GAA GAA GAT GTA TAC TGG GGA GC - #A GAA ACC GAA TGG CTG 
624 
Trp Glu Pro Glu Glu Asp Val Tyr Trp Gly Al - #a Glu Thr Glu Trp Leu 
# 205 
- GGA GAC AAG CGC TAT GAA GGT GAC CGA GAG CT - #C GAA AAT CCC CTG GGA 
672 
Gly Asp Lys Arg Tyr Glu Gly Asp Arg Glu Le - #u Glu Asn Pro Leu Gly 
# 220 
- GCC GTA CAA ATG GGA CTC ATC TAT GTA AAC CC - #C GAA GGA CCC AAC GGC 
720 
Ala Val Gln Met Gly Leu Ile Tyr Val Asn Pr - #o Glu Gly Pro Asn Gly 
225 2 - #30 2 - #35 2 - 
#40 
- AAG CCA GAC CCT ATC GCT GCT GCG CGT GAT AT - #T CGT GAG ACT TTT GGC 
768 
Lys Pro Asp Pro Ile Ala Ala Ala Arg Asp Il - #e Arg Glu Thr Phe Gly 
# 255 
- CGA ATG GCA ATG AAT GAC GAA GAA ACC GTG GC - #T CTC ATA GCG GGT GGA 
816 
Arg Met Ala Met Asn Asp Glu Glu Thr Val Al - #a Leu Ile Ala Gly Gly 
# 270 
- CAC ACC TTC GGA AAA ACC CAT GGT GCT GCC GA - #T GCG GAG AAA TAT GTG 
864 
His Thr Phe Gly Lys Thr His Gly Ala Ala As - #p Ala Glu Lys Tyr Val 
# 285 
- GGC CGA GAG CCT GCC GCC GCA GGT ATT GAA GA - #A ATG AGC CTG GGG TGG 
912 
Gly Arg Glu Pro Ala Ala Ala Gly Ile Glu Gl - #u Met Ser Leu Gly Trp 
# 300 
- AAA AAC ACC TAC GGC ACC GGA CAC GGT GCG GA - #T ACC ATC ACC AGT GGA 
960 
Lys Asn Thr Tyr Gly Thr Gly His Gly Ala As - #p Thr Ile Thr Ser Gly 
305 3 - #10 3 - #15 3 - 
#20 
- CTA GAA GGC GCC TGG ACC AAG ACC CCT ACT CA - #A TGG AGC AAT AAC TTT 
1008 
Leu Glu Gly Ala Trp Thr Lys Thr Pro Thr Gl - #n Trp Ser Asn Asn Phe 
# 335 
- TTT GAA AAC CTC TTT GGT TAC GAG TGG GAG CT - #T ACC AAA AGT CCA GCT 
1056 
Phe Glu Asn Leu Phe Gly Tyr Glu Trp Glu Le - #u Thr Lys Ser Pro Ala 
# 350 
- GGA GCT TAT CAG TGG AAA CCA AAA GAC GGT GC - #C GGG GCT GGC ACC ATA 
1104 
Gly Ala Tyr Gln Trp Lys Pro Lys Asp Gly Al - #a Gly Ala Gly Thr Ile 
# 365 
- CCG GAT GCA CAT GAT CCC AGC AAG TCG CAC GC - #T CCA TTT ATG CTC ACT 
1152 
Pro Asp Ala His Asp Pro Ser Lys Ser His Al - #a Pro Phe Met Leu Thr 
# 380 
- ACG GAC CTG GCG CTG CGC ATG GAC CCT GAT TA - #C GAA AAA ATT TCT CGA 
1200 
Thr Asp Leu Ala Leu Arg Met Asp Pro Asp Ty - #r Glu Lys Ile Ser Arg 
385 3 - #90 3 - #95 4 - 
#00 
- CGG TAC TAT GAA AAC CCT GAT GAG TTT GCA GA - #T GCT TTC GCG AAA GCA 
1248 
Arg Tyr Tyr Glu Asn Pro Asp Glu Phe Ala As - #p Ala Phe Ala Lys Ala 
# 415 
- TGG TAC AAA CTG ACA CAC AGA GAT ATG GGA CC - #A AAG GTG CGC TAC CTG 
1296 
Trp Tyr Lys Leu Thr His Arg Asp Met Gly Pr - #o Lys Val Arg Tyr Leu 
# 430 
- GGA CCA GAA GTG CCT CAG GAA GAC CTC ATC TG - #G CAA GAC CCT ATA CCA 
1344 
Gly Pro Glu Val Pro Gln Glu Asp Leu Ile Tr - #p Gln Asp Pro Ile Pro 
# 445 
- GAT GTA AGC CAT CCT CTT GTA GAC GAA AAC GA - #T ATT GAA GGC CTA AAA 
1392 
Asp Val Ser His Pro Leu Val Asp Glu Asn As - #p Ile Glu Gly Leu Lys 
# 460 
- GCC AAA ATC CTG GAA TCG GGA CTG ACG GTA AG - #C GAG CTG GTA AGC ACG 
1440 
Ala Lys Ile Leu Glu Ser Gly Leu Thr Val Se - #r Glu Leu Val Ser Thr 
465 4 - #70 4 - #75 4 - 
#80 
- GCA TGG GCT TCT GCA TCT ACT TTT AGA AAC TC - #T GAC AAG CGC GGC GGT 
1488 
Ala Trp Ala Ser Ala Ser Thr Phe Arg Asn Se - #r Asp Lys Arg Gly Gly 
# 495 
- GCC AAC GGT GCA CGT ATA CGA CTG GCC CCA CA - #A AAA GAC TGG GAA GTA 
1536 
Ala Asn Gly Ala Arg Ile Arg Leu Ala Pro Gl - #n Lys Asp Trp Glu Val 
# 510 
- AAC AAC CCT CAG CAA CTT GCC AGG GTA CTC AA - #A ACA CTA GAA GGT ATC 
1584 
Asn Asn Pro Gln Gln Leu Ala Arg Val Leu Ly - #s Thr Leu Glu Gly Ile 
# 525 
- CAG GAG GAC TTT AAC CAG GCG CAA TCA GAT AA - #C AAA GCA GTA TCG TTG 
1632 
Gln Glu Asp Phe Asn Gln Ala Gln Ser Asp As - #n Lys Ala Val Ser Leu 
# 540 
- GCC GAC CTG ATT GTG CTG GCC GGC TGT GCG GG - #T GTA GAA AAA GCT GCA 
1680 
Ala Asp Leu Ile Val Leu Ala Gly Cys Ala Gl - #y Val Glu Lys Ala Ala 
545 5 - #50 5 - #55 5 - 
#60 
- AAA GAT GCT GGC CAT GAG GTG CAG GTG CCT TT - #C AAC CCG GGA CGA GCG 
1728 
Lys Asp Ala Gly His Glu Val Gln Val Pro Ph - #e Asn Pro Gly Arg Ala 
# 575 
- GAT GCC ACC GCT GAG CAA ACC GAT GTG GAA GC - #T TTC GAA GCA CTA GAG 
1776 
Asp Ala Thr Ala Glu Gln Thr Asp Val Glu Al - #a Phe Glu Ala Leu Glu 
# 590 
- CCA GCG GCT GAC GGC TTT AGA AAC TAC ATT AA - #A CCG GAG CAT AAA GTA 
1824 
Pro Ala Ala Asp Gly Phe Arg Asn Tyr Ile Ly - #s Pro Glu His Lys Val 
# 605 
- TCC GCT GAG GAA ATG CTC GTA GAC CGG GCG CA - #G CTT CTG TCG CTT TCG 
1872 
Ser Ala Glu Glu Met Leu Val Asp Arg Ala Gl - #n Leu Leu Ser Leu Ser 
# 620 
- GCA CCA GAA ATG ACT GCT TTG GTA GGC GGT AT - #G CGT GTA CTG GGC ACC 
1920 
Ala Pro Glu Met Thr Ala Leu Val Gly Gly Me - #t Arg Val Leu Gly Thr 
625 6 - #30 6 - #35 6 - 
#40 
- AAC TAC GAC GGT TCG CAG CAT GGA GTG TTT AC - #A AAT AAG CCG GGT CAG 
1968 
Asn Tyr Asp Gly Ser Gln His Gly Val Phe Th - #r Asn Lys Pro Gly Gln 
# 655 
- CTA TCC AAT GAC TTC TTT GTA AAC CTG CTA GA - #C CTC AAC ACT AAA TGG 
2016 
Leu Ser Asn Asp Phe Phe Val Asn Leu Leu As - #p Leu Asn Thr Lys Trp 
# 670 
- CGA GCC AGC GAT GAA TCA GAC AAA GTT TTT GA - #A GGC AGA GAC TTC AAA 
2064 
Arg Ala Ser Asp Glu Ser Asp Lys Val Phe Gl - #u Gly Arg Asp Phe Lys 
# 685 
- ACT GGC GAA GTA AAG TGG AGT GGC ACC CGG GT - #A GAC CTG ATC TTC GGA 
2112 
Thr Gly Glu Val Lys Trp Ser Gly Thr Arg Va - #l Asp Leu Ile Phe Gly 
# 700 
- TCC AAT TCC GAG CTA AGA GCC CTC GCA GAA GT - #G TAC GGC TGT GCA GAT 
2160 
Ser Asn Ser Glu Leu Arg Ala Leu Ala Glu Va - #l Tyr Gly Cys Ala Asp 
705 7 - #10 7 - #15 7 - 
#20 
- TCT GAA GAA AAG TTT GTT AAA GAT TTT GTG AA - #G GCC TGG GCC AAA GTA 
2208 
Ser Glu Glu Lys Phe Val Lys Asp Phe Val Ly - #s Ala Trp Ala Lys Val 
# 735 
# 2238 GG TTT GAT CTG AAA TAA 
Met Asp Leu Asp Arg Phe Asp Leu Lys 
# 745 
- (2) INFORMATION FOR SEQ ID NO:8: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 745 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (v) FRAGMENT TYPE: internal 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- Met Glu Asn His Lys His Ser Gly Ser Ser Th - #r Tyr Asn Thr Asn Thr 
# 15 
- Gly Gly Lys Cys Pro Phe Thr Gly Gly Ser Le - #u Lys Gln Ser Ala Gly 
# 30 
- Gly Gly Thr Lys Asn Arg Asp Trp Trp Pro As - #n Met Leu Asn Leu Gly 
# 45 
- Ile Leu Arg Gln His Ser Ser Leu Ser Asp Pr - #o Asn Asp Pro Asp Phe 
# 60 
- Asp Tyr Ala Glu Glu Phe Lys Lys Leu Asp Le - #u Ala Ala Val Lys Lys 
# 80 
- Asp Leu Ala Ala Leu Met Thr Asp Ser Gln As - #p Trp Trp Pro Ala Asp 
# 95 
- Tyr Gly His Tyr Gly Pro Phe Phe Ile Arg Me - #t Ala Trp His Ser Ala 
# 110 
- Gly Thr Tyr Arg Ile Gly Asp Gly Arg Gly Gl - #y Gly Gly Ser Gly Ser 
# 125 
- Gln Arg Phe Ala Pro Leu Asn Ser Trp Pro As - #p Asn Ala Asn Leu Asp 
# 140 
- Lys Ala Arg Leu Leu Leu Trp Pro Ile Lys Gl - #n Lys Tyr Gly Arg Lys 
145 1 - #50 1 - #55 1 - 
#60 
- Ile Ser Trp Ala Asp Leu Met Ile Leu Thr Gl - #y Asn Val Ala Leu Glu 
# 175 
- Thr Met Gly Phe Lys Thr Phe Gly Phe Ala Gl - #y Gly Arg Ala Asp Val 
# 190 
- Trp Glu Pro Glu Glu Asp Val Tyr Trp Gly Al - #a Glu Thr Glu Trp Leu 
# 205 
- Gly Asp Lys Arg Tyr Glu Gly Asp Arg Glu Le - #u Glu Asn Pro Leu Gly 
# 220 
- Ala Val Gln Met Gly Leu Ile Tyr Val Asn Pr - #o Glu Gly Pro Asn Gly 
225 2 - #30 2 - #35 2 - 
#40 
- Lys Pro Asp Pro Ile Ala Ala Ala Arg Asp Il - #e Arg Glu Thr Phe Gly 
# 255 
- Arg Met Ala Met Asn Asp Glu Glu Thr Val Al - #a Leu Ile Ala Gly Gly 
# 270 
- His Thr Phe Gly Lys Thr His Gly Ala Ala As - #p Ala Glu Lys Tyr Val 
# 285 
- Gly Arg Glu Pro Ala Ala Ala Gly Ile Glu Gl - #u Met Ser Leu Gly Trp 
# 300 
- Lys Asn Thr Tyr Gly Thr Gly His Gly Ala As - #p Thr Ile Thr Ser Gly 
305 3 - #10 3 - #15 3 - 
#20 
- Leu Glu Gly Ala Trp Thr Lys Thr Pro Thr Gl - #n Trp Ser Asn Asn Phe 
# 335 
- Phe Glu Asn Leu Phe Gly Tyr Glu Trp Glu Le - #u Thr Lys Ser Pro Ala 
# 350 
- Gly Ala Tyr Gln Trp Lys Pro Lys Asp Gly Al - #a Gly Ala Gly Thr Ile 
# 365 
- Pro Asp Ala His Asp Pro Ser Lys Ser His Al - #a Pro Phe Met Leu Thr 
# 380 
- Thr Asp Leu Ala Leu Arg Met Asp Pro Asp Ty - #r Glu Lys Ile Ser Arg 
385 3 - #90 3 - #95 4 - 
#00 
- Arg Tyr Tyr Glu Asn Pro Asp Glu Phe Ala As - #p Ala Phe Ala Lys Ala 
# 415 
- Trp Tyr Lys Leu Thr His Arg Asp Met Gly Pr - #o Lys Val Arg Tyr Leu 
# 430 
- Gly Pro Glu Val Pro Gln Glu Asp Leu Ile Tr - #p Gln Asp Pro Ile Pro 
# 445 
- Asp Val Ser His Pro Leu Val Asp Glu Asn As - #p Ile Glu Gly Leu Lys 
# 460 
- Ala Lys Ile Leu Glu Ser Gly Leu Thr Val Se - #r Glu Leu Val Ser Thr 
465 4 - #70 4 - #75 4 - 
#80 
- Ala Trp Ala Ser Ala Ser Thr Phe Arg Asn Se - #r Asp Lys Arg Gly Gly 
# 495 
- Ala Asn Gly Ala Arg Ile Arg Leu Ala Pro Gl - #n Lys Asp Trp Glu Val 
# 510 
- Asn Asn Pro Gln Gln Leu Ala Arg Val Leu Ly - #s Thr Leu Glu Gly Ile 
# 525 
- Gln Glu Asp Phe Asn Gln Ala Gln Ser Asp As - #n Lys Ala Val Ser Leu 
# 540 
- Ala Asp Leu Ile Val Leu Ala Gly Cys Ala Gl - #y Val Glu Lys Ala Ala 
545 5 - #50 5 - #55 5 - 
#60 
- Lys Asp Ala Gly His Glu Val Gln Val Pro Ph - #e Asn Pro Gly Arg Ala 
# 575 
- Asp Ala Thr Ala Glu Gln Thr Asp Val Glu Al - #a Phe Glu Ala Leu Glu 
# 590 
- Pro Ala Ala Asp Gly Phe Arg Asn Tyr Ile Ly - #s Pro Glu His Lys Val 
# 605 
- Ser Ala Glu Glu Met Leu Val Asp Arg Ala Gl - #n Leu Leu Ser Leu Ser 
# 620 
- Ala Pro Glu Met Thr Ala Leu Val Gly Gly Me - #t Arg Val Leu Gly Thr 
625 6 - #30 6 - #35 6 - 
#40 
- Asn Tyr Asp Gly Ser Gln His Gly Val Phe Th - #r Asn Lys Pro Gly Gln 
# 655 
- Leu Ser Asn Asp Phe Phe Val Asn Leu Leu As - #p Leu Asn Thr Lys Trp 
# 670 
- Arg Ala Ser Asp Glu Ser Asp Lys Val Phe Gl - #u Gly Arg Asp Phe Lys 
# 685 
- Thr Gly Glu Val Lys Trp Ser Gly Thr Arg Va - #l Asp Leu Ile Phe Gly 
# 700 
- Ser Asn Ser Glu Leu Arg Ala Leu Ala Glu Va - #l Tyr Gly Cys Ala Asp 
705 7 - #10 7 - #15 7 - 
#20 
- Ser Glu Glu Lys Phe Val Lys Asp Phe Val Ly - #s Ala Trp Ala Lys Val 
# 735 
- Met Asp Leu Asp Arg Phe Asp Leu Lys 
# 745 
__________________________________________________________________________