A purified thermostable enzyme is derived from the archael bacterium AEPII1a. The enzyme has a molecular weight of about 60.9 kilodaltons and has cellulase activity. The enzyme can be produced from native or recombinant host cells and can be used to aid in the digestion of cellulose where desired.

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 polypeptide of 
the present invention has been putatively identified as an endoglucanase 
and in particular an enzyme having carboxymethyl cellulase activity. 
Cellulose, a fibrous, tough, water-insoluble substance is found in the cell 
walls of plants, particularly, in stalks, stems, trunks and all the woody 
portions of plant tissues. Cellulose constitutes much of the mass of wood, 
and cotton is almost pure cellulose. Because cellulose is a linear, 
unbranched homopolysaccharide of 10,000 to 15,000 D-glucose units, it 
resembles amylose and the main chains of glycogen. But there is a very 
important difference, in cellulose, the glucose residues have the beta 
configuration, whereas in amylose, amylopectin and glycogen, the glucose 
is in the alpha configuration. The glucose residues in cellulose are 
linked by (beta 1.fwdarw.4) glycosidic bonds. This difference gives 
cellulose and amylose very different 3-dimensional structures and physical 
properties. 
Cellulose cannot be used by most animals as a source of stored fuel, 
because the (beta 1.fwdarw.4) linkages of cellulose are not hydrolyzed by 
alpha-amylases. Termites readily digest cellulose but only because their 
intestinal tract harbors a symbiotic microorganism, trichonympha, which 
secretes cellulase, an enzyme that hydrolyses (beta 1.fwdarw.4) linkages 
between glucose units. The only vertebrates able to use cellulose as food 
are cattle and other ruminant animals (sheep, goats, camels and giraffes). 
The extra stomachs "rumens" of these animals teem with bacteria and 
protists that secrete cellulase. 
The enzymatic hydrolysis of cellulose is considered to require the action 
of both endoglucanases (1,4-beta-D-glucan glucanohydrolase) and 
exoglucanases (1,4-beta-D-glucan cellobiohydrolase). A synergistic 
interaction of these enzymes is necessary for the complete hydrolysis of 
crystalline cellulose. (Caughlin, M. P., Genet. Eng. Rev., 3:39-109 
(1985)). For the complete degradation of cellulose (cellulose to glucose), 
.beta.-glucosidase might be required if the "exo" enzyme does not release 
glucose. 1,4-.beta.-D-glucan glucohydrolase is another type of "exo" 
cellulase. 
Thermophilic bacteria have received considerable attention as sources of 
highly active and thermostable cellulolytic and xylanolytic enzymes 
(Bronneomeier, K. and Staudenbauer, W. L., D. R. Woods (Ed.), The 
Clostridia and Biotechnology, Butterworth Publishers, Stoneham, Mass. 
(1993). Recently, the most extremely thermophilic organotrophic eubacteria 
presently known have been isolated and characterized. These bacteria, 
which belong to the genus thermotoga, are fermentative microorganisms 
metabolizing a variety of carbohydrates (Huber, R. and Stetter, K. O., in 
Ballows, et al., (Ed.), The Procaryotes, 2nd Ed., Springer-Verlaz, New 
York, pgs. 3809-3819 (1992)). 
In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation of the 
bacterium Thermotoga maritima is described. T. maritima is a eubacterium 
that is strictly anaerobic, rod-shaped, fermentative, hyperthermophilic, 
and grows between 55.degree. C. and 90.degree. C., with an optimum growth 
temperature of about 80.degree. C. This eubacterium has been isolated from 
geothermally heated sea floors in Italy and the Azores. T. maritima cells 
have a sheath-like structure and monotrichous flagellation. T. maritima is 
classified in the eubacterium kingdom by virtue of having murein and fatty 
acid-containing lipids, diphtheria-toxin-resistant elongation factor 2, an 
RNA polymerase subunit pattern, and sensitivity to antibiotics. 
Because to date most organisms identified from the archaeal domain are 
thermophiles or hyperthermophiles, archaeal bacteria are also considered a 
fertile source of thermophilic enzymes. 
The polynucleotide and polypeptide encoded thereby of the present invention 
has been putatively identified as an endoglucanase enzyme having 
carboxymethyl cellulase activity. 
In accordance with one aspect of the present invention, there is provided a 
novel enzyme, 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 an enzyme of the present 
invention including mRNAs, DNAs, cDNAs, genomic DNAs as well as active 
analogs and fragments of such enzymes. 
In accordance with another aspect of the present invention there are 
provided isolated nucleic acid molecules encoding mature polypeptides 
expressed by the DNA contained in ATCC Deposit No. 97516. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for producing such polypeptide by recombinant 
techniques comprising culturing recombinant prokaryotic and/or eukaryotic 
host cells, containing a nucleic acid sequence encoding an enzyme of the 
present invention, under conditions promoting expression of said enzyme 
and subsequent recovery of said enzyme. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such enzyme, or polynucleotide encoding 
such enzyme for degradation of cellulose for the conversion of plant 
biomass into fuels and chemicals, for use in detergents, the textile 
industry, in animal feed, in waste treatment, and in the fruit 
juice/brewing industry for the clarification and extraction of juices. 
In accordance with yet a further aspect of the present invention, there is 
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. 
These and other aspects of the present invention should be apparent to 
those skilled in the art from the teachings herein.

The term "gene" means the segment of DNA involved in producing 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 are 
ultimately processed 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. 
A "promotor sequence" is a DNA regulatory region capable of binding RNA 
polymerase in a cell and initiating transcription of a downstream (3' 
direction) coding sequence. The promoter is part of the DNA sequence. This 
sequence region has a start codon at its 3' terminus. The promoter 
sequence does include the minimum number of bases where elements necessary 
to initiate transcription at levels detectable above background. However, 
after the RNA polymerase binds the sequence and transcription is initiated 
at the start codon (3' terminus with a promoter), transcription proceeds 
downstream in the 3' direction. Within the promotor sequence will be found 
a transcription initiation site (conveniently defined by mapping with 
nuclease S1) as well as protein binding domains (consensus sequences) 
responsible for the binding of RNA polymerase. 
The present invention provides a purified thermostable enzyme that 
catalyzes the hydrolysis of the beta 1,4 glycosidic bonds in cellulose to 
thereby degrade cellulose. The purified enzyme is an endoglucanase derived 
from an organism referred herein as "AEPII1a" which is a thermophilic 
archaeal bacteria which has a very high temperature optimum. The organism 
is strictly anaerobic, rod-shaped and fermentative, and grows between 
55.degree. and 90.degree. C. (optimally at 85.degree. C.). AEPII1a was 
discovered in a shallow marine hydrothermal area in Vulcano, Italy. The 
organism has coccoid cells occurring in singlets or pairs. AEPII1a grows 
optimally at 85.degree. C. and pH 6.5 in a marine medium with cellulose as 
a substrate and nitrogen in gas phase. 
The polynucleotide of this invention was originally recovered from a 
genomic gene library derived from AEPII1a as described below. It contains 
an open reading frame encoding a protein of 553 amino acid residues. 
In a preferred embodiment, the endoglucanase enzyme of the present 
invention has a molecular weight of about 60.9 kilodaltons as measured by 
SDS-PAGE gel electrophoresis and an inferred molecular weight from the 
nucleotide sequence of the gene. This purified enzyme may be used to 
catalyze the enzymatic degradation of cellulose where desired. The 
endoglucanase enzyme of the present invention has a very high 
thermostability and has the closest homology to endo-1,4-beta-glucanase 
from Xanthomonas campestris with 50% identity and 71% similarity at the 
amino acid level. 
In accordance with an aspect of the present invention, there are provided 
isolated nucleic acid molecules (polynucleotides) which encode for the 
mature enzyme having the deduced amino acid sequence of FIG. 1 (SEQ ID 
NO:2). 
In accordance with another aspect of the present invention, there is 
provided an isolated polynucleotide encoding the enzyme of the present 
invention which has been deposited with an appropriate depository for the 
deposit of biological material. The deposited material is a pQET (Qiagen, 
Inc.) plasmid comprising the DNA of FIG. 1. The deposit has been deposited 
with the American Type Culture Collection, 12301 Parklawn Drive, 
Rockville, Md. 20852, USA, on Apr. 22, 1996 and assigned ATCC Deposit No. 
97516. 
The deposit has been made under the terms of the Budapest Treaty on the 
International Recognition of the deposit of micro-organisms for purposes 
of patent procedure. The strain will be irrevocably and without 
restriction or condition released to the public upon the issuance of a 
patent. The deposit is provided merely as convenience to those of skill in 
the art and are not an admission that a deposit be required under 35 
U.S.C. .sctn.112. The sequences of the polynucleotides contained in the 
deposited materials, as well as the amino acid sequences of the 
polypeptides encoded thereby, are controlling in the event of any conflict 
with any description of sequences herein. A license may be required to 
make, use or sell the deposited materials, and no such license is hereby 
granted. 
This invention, in addition to the isolated nucleic acid molecule encoding 
an endoglucanase enzyme disclosed in FIG. 1 (SEQ ID NO:1), also provides 
substantially similar sequences. Isolated nucleic acid sequences are 
substantially similar if: (i) they are capable of hybridizing under 
stringent conditions, hereinafter described, to SEQ ID NO:1; or (ii) they 
encode DNA sequences which are degenerate to SEQ ID NO:1. Degenerate DNA 
sequences encode the amino acid sequence of SEQ ID NO:2, but have 
variations in the nucleotide coding sequences. As used herein, 
"substantially similar" refers to the sequences having similar identity to 
the sequences of the instant invention. The nucleotide sequences that are 
substantially similar can be identified by hybridization or by sequence 
comparison. Enzyme sequences that are substantially similar can be 
identified by one or more of the following: proteolytic digestion, gel 
electrophoresis and/or microsequencing. 
One means for isolating a nucleic acid molecule encoding an endoglucanase 
enzyme is to probe a genomic 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 to one skilled in the art that SEQ ID NO:1, or 
fragments thereof (comprising at least 15 contiguous nucleotides), is a 
particularly useful probe. Other particular useful probes for this purpose 
are hybridizable fragments to the sequences of SEQ ID NO:1 (i.e., 
comprising at least 15 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 acid is first 
prehybridized for 30 minutes at 45.degree. C. in a solution consisting of 
0.9M 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-10.degree. C. for the 
oligo-nucleotide 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. See J. Sambrook et al., 
Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor 
Laboratory) which is hereby incorporated by reference in its entirety. 
"Identity" as the term is used herein, refers to a polynucleotide sequence 
which comprises a percentage of the same bases as a reference 
polynucleotide (SEQ ID NO:1). For example, a polynucleotide which is at 
least 90% identical to a reference polynucleotide, has polynucleotide 
bases which are identical in 90% of the bases which make up the reference 
polynucleotide and may have different bases in 10% of the bases which 
comprise that polynucleotide sequence. 
The present invention also relates to polynucleotides which differ from the 
reference polynucleotide such that the changes are silent changes, for 
example the changes do not alter the amino acid sequence encoded by the 
polynucleotide. The present invention also relates to nucleotide changes 
which result in amino acid substitutions, additions, deletions, fusions 
and truncations in the enzyme encoded by the reference polynucleotide (SEQ 
ID NO:1). In a preferred aspect of the invention these enzymes retain the 
same biological action as the enzyme encoded by the reference 
polynucleotide. 
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 animal sources or to screen such sources for 
related sequences. 
The coding sequence for the endoglucanase enzyme of the present invention 
was identified by preparing an AEPII1a genomic DNA library and screening 
the library for the clones having endoglucanase activity. Such methods for 
constructing a genomic gene library are well-known in the art. One means, 
for example, comprises shearing DNA isolated from AEPII1a by physical 
disruption. A small amount of the sheared DNA is checked on an agarose gel 
to verify that the majority of the DNA is in the desired size range 
(approximately 3-6 kb). The DNA is then blunt ended using Mung Bean 
Nuclease, incubated at 37.degree. C. and phenol/chloroform extracted. The 
DNA is then methylated using Eco RI Methylase. Eco RI linkers are then 
ligated to the blunt ends through the use of T4 DNA ligase and incubation 
at 4.degree. C. The ligation reaction is then terminated and the DNA is 
cut-back with Eco RI restriction enzyme. The DNA is then size fractionated 
on a sucrose gradient following procedures known in the art, for example, 
Maniatis, T., et al., Molecular Cloning, Cold Spring Harbor Press, New 
York, 1982, which is hereby incorporated by reference in its entirety. 
A plate assay is then performed to get an approximate concentration of the 
DNA. Ligation reactions are then performed and 1 .mu.l of the ligation 
reaction is packaged to construct a library. Packaging, for example, may 
occur through the use of purified .lambda.gt11 phage arms cut with EcoRI 
and DNA cut with EcoRI after attaching EcoRI linkers. The DNA and 
.lambda.gt11 arms are ligated with DNA ligase. The ligated DNA is then 
packaged into infectious phage particles. The packaged phages are used to 
infect E. coli cultures and the infected cells are spread on agar plates 
to yield plates carrying thousands of individual phage plaques. The 
library is then amplified. 
In a preferred embodiment, the enzyme of the present invention, was 
isolated from an AEPII1a library by the following technique: 
1. .lambda.gt11 AEPII1a library was plated onto 6 LB/GelRite/0.1% CMC/NZY 
agar plates (.about.4,800 plaque forming units/plate) in E.coli Y1090 host 
with LB agarose containing 1 mM IPTG as top agarose. The plates were 
incubated at 37.degree. C. overnight. 
2. Plates were chilled at 4.degree. C. for one hour. 
3. The plates were overlayed with Duralon membranes (Stratagene) at room 
temperature for one hour and the membranes were oriented and lifted off 
the plates and stored at 4.degree. C. 
4. The top agarose layer was removed and plates were incubated at 
72.degree. C. for .about.3 hours. 
5. The plate surface was rinsed with NaCl. 
6. The plate was stained with 0.1% Congo Red for 15 minutes. 
7. The plate was destained with 1M NaCl. 
8. The putative positives identified on plate were isolated from the 
Duralon membrane (positives are identified by clearing zones around 
clones). The phage was eluted from the membrane by incubating in 500 .mu.l 
SM+25 .mu.l CHCl.sub.3 to elute. 
9. Insert DNA was subcloned into pBluescript II SK(+) cloning vector 
(Stratagene), and subclones were reassayed for CMCase activity using the 
following protocol: 
i) Spin 1 ml overnight miniprep of clone at maximum speed for 3 minutes. 
ii) Decant the supernatant and use it to fill "wells" that have been made 
in an LB/GelRite/0.1% CMC plate. 
iii) Incubate at 72.degree. C. for 2 hours. 
iv) Stain with 0.1% Congo Red for 15 minutes. 
v) Destain with 1M NaCl for 15 minutes. 
vi) Identify positives by clearing zone around clone. 
Fragments of the full length gene of the present invention may be used as a 
hybridization probe 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 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. 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. 
The isolated nucleic acid sequences and other enzymes may then be measured 
for retention of biological activity characteristic to the enzyme of the 
present invention, for example, in an assay for detecting enzymatic 
endoglucanase activity. Such enzymes include truncated forms of 
endoglucanase, and variants such as deletion and insertion variants. 
Examples of such assays include an assay for the detection of endoglucanase 
activity based on specific interaction of direct dyes such as Congo red 
with polysaccharides. This colorant reacts with beta-1,4-glucans causing a 
visible red shift (Wood, P. J., Carbohydr. Res., 85:271 (1980) and Wood, 
P. J., Carbohydr. Res., 94:c19 (1981)). The preferred substrate for the 
test is carboxymethylcellulose (CMC) which can be obtained from different 
sources (Hercules Inc., Wilmington, Del., Type 4M6F or Sigma Chemical 
Company, St. Louis, Mo., Medium Viscosity). The CMC is incorporated as the 
main carbon sources into a minimal agar medium in quantities of 0.1-1.0%. 
The microorganisms can be screened directly on these plates, but the 
replica plating technique from a master plate is preferable since the 
visualization of the activity requires successive flooding with the 
reagents, which would render the reisolation of active colonies difficult. 
Such endoglucanase-producing colonies are detectable after a suitable 
incubation time (1-3 days depending on the growth), by flooding the plate 
with 10 ml of a 0.1% aqueous solution of Congo Red. The coloration is 
terminated after 20 minutes by pouring off the dye and flooding the plates 
with 10 ml of 5M NaCl solution (commercial salt can be used). After an 
additional 20 minutes, the salt solution is discarded and endoglucanase 
activity is revealed by a pale-orange zone around the active 
microorganisms. In some cases, these zones can be enhanced by treating the 
plates with 1N acetic acid, causing the dye to change its color to blue. 
The same technique can be used as a cup-plate diffusion assay with 
excellent sensitivity for the determination of endoglucanase activity in 
culture filtrates or during enzyme purification steps (Carger, J. H., 
Anal. Biochem., 153:75 (1986)). See generally, Methods for Measuring 
Cellulase Activities, Methods in Enzymology, Vol. 160, pgs. 87-116. 
The enzyme of the present invention has enzymatic activity with respect to 
the hydrolysis of the beta 1,4 glycosidic bonds in carboxymethylcellulose, 
since the halos discussed above are shown around the colonies. 
The polynucleotide of the present invention may be in the form of 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 sequence which 
encodes the mature enzyme may be identical to the coding sequence shown in 
FIG. 1 (SEQ ID NO:1) and/or that of the deposited clone 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 
enzyme as the DNA of FIG. 1 (SEQ ID NO:1). 
The polynucleotide which encodes for the mature enzyme of FIG. 1 (SEQ ID 
NO:2) 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 enzyme having the deduced amino acid sequence of FIG. 1 
(SEQ ID NO:2). 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 enzyme as shown in FIG. 1 (SEQ ID NO:2) as well as variants of such 
polynucleotides which variants encode for a fragment, derivative or analog 
of the enzyme of FIG. 1 (SEQ ID NO:2). Such nucleotide variants include 
deletion variants, substitution variants and addition or insertion 
variants. 
As hereinabove indicated, the polynucleotide may have a coding sequence 
which is a naturally occurring allelic variant of the coding sequence 
shown in FIG. 1 (SEQ ID NO:1). 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. 
The present invention also includes polynucleotides, wherein the coding 
sequence for the mature enzyme may be fused in the same reading frame to a 
polynucleotide sequence which aids in expression and secretion of an 
enzyme from a host cell, for example, a leader sequence which functions to 
control transport of an enzyme from the cell. The enzyme having a leader 
sequence is a preprotein and may have the leader sequence cleaved by the 
host cell to form the mature form of the enzyme. The polynucleotides may 
also encode for a proprotein which is the mature protein plus additional 
5' amino acid residues. A mature protein having a prosequence is a 
proprotein and is an inactive form of the protein. Once the prosequence is 
cleaved an active mature protein remains. 
Thus, for example, the polynucleotide of the present invention may encode 
for a mature enzyme, or for an enzyme having a prosequence or for an 
enzyme having both a prosequence and a presequence (leader sequence). 
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. 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 
FIG. 1 (SEQ ID NO:1). 
Alternatively, the polynucleotide may have at least 15 bases, preferably at 
least 30 bases, and more preferably at least 50 bases which hybridize to 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 
polynucleotide of SEQ ID NO:1, for example, for recovery of the 
polynucleotide 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 enzyme of SEQ 
ID NO:2 as well as fragments thereof, which fragments have at least 30 
bases and preferably at least 50 bases and to enzymes encoded by such 
polynucleotides. 
The present invention further relates to a enzyme which has the deduced 
amino acid sequence of FIG. 1 (SEQ ID NO:2), as well as fragments, analogs 
and derivatives of such enzyme. 
The terms "fragment," "derivative" and "analog" when referring to the 
enzyme of FIG. 1 (SEQ ID NO:2) means a enzyme which retains essentially 
the same biological function or activity as such enzyme. Thus, an analog 
includes a proprotein which can be activated by cleavage of the proprotein 
portion to produce an active mature enzyme. 
The enzyme 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 enzyme of FIG. 1 (SEQ ID NO:2) 
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 term "isolated" means that the material is removed from its original 
environment (e.g., the natural environment if it is naturally occurring). 
For example, a naturally-occurring polynucleotide or enzyme present in a 
living animal is not isolated, but the same polynucleotide or enzyme, 
separated from some or all of the coexisting materials in the natural 
system, is isolated. Such polynucleotides could be part of a vector and/or 
such polynucleotides or enzymes could be part of a composition, and still 
be isolated in that such vector or composition is not part of its natural 
environment. 
The enzymes of the present invention include the enzyme of SEQ ID NO:2 (in 
particular the mature enzyme) as well as enzymes which have at least 70% 
similarity (preferably at least 70% identity) to the enzyme of SEQ ID NO:2 
and more preferably at least 90% similarity (more preferably at least 90% 
identity) to the enzyme of SEQ ID NO:2 and still more preferably at least 
95% similarity (still more preferably at least 95% identity) to the enzyme 
of SEQ ID NO:2 and also include portions of such enzymes with such portion 
of the enzyme generally containing at least 30 amino acids and more 
preferably at least 50 amino acids. 
As known in the art "similarity" between two enzymes is determined by 
comparing the amino acid sequence and its conserved amino acid substitutes 
of one enzyme to the sequence of a second enzyme. Similarity may be 
determined by procedures which are well-known in the art, for example, a 
BLAST program (Basic Local Alignment Search Tool at the National Center 
for Biological Information). 
A variant, i.e. a "fragment", "analog" or "derivative" enzyme, and 
reference enzyme may differ in amino acid sequence by one or more 
substitutions, additions, deletions, fusions and truncations, which may be 
present in any combination. 
Among preferred variants are those that vary from a reference by 
conservative amino acid substitutions. Such substitutions are those that 
substitute a given amino acid in a polypeptide by another amino acid of 
like characteristics. Typically seen as conservative substitutions are the 
replacements, one for another, among the aliphatic amino acids Ala, Val, 
Leu and Ile; interchange of the hydroxyl residues Ser and Thr, exchange of 
the acidic residues Asp and Glu, substitution between the amide residues 
Asn and Gln, exchange of the basic residues Lys and Arg and replacements 
among the aromatic residues Phe, Tyr. 
Most highly preferred are variants which retain the same biological 
function and activity as the reference polypeptide from which it varies. 
Fragments or portions of the enzymes of the present invention may be 
employed for producing the corresponding full-length enzyme by peptide 
synthesis; therefore, the fragments may be employed as intermediates for 
producing the full-length enzymes. Fragments or portions of the 
polynucleotides of the present invention may be used to synthesize 
full-length polynucleotides of the present invention. 
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 containing the polynucleotides of this 
invention. Such vectors may be, for example, a cloning vector or an 
expression vector. The vector may be, for example, in the form of a 
plasmid, a viral particle, 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 
(Stratagene); pTRC99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic: 
pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVLSV40 (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 GEM1 (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. 
The enzyme of this invention may be employed for any purpose in which such 
enzyme activity is necessary or desired. In a preferred embodiment the 
enzyme is employed for catalyzing the hydrolysis of cellulose. The 
degradation of cellulose may be used for the conversion of plant biomass 
into fuels and chemicals. 
The enzyme of the present invention may also be employed in the detergent 
and textile industry, in the production of animal feed, in waste treatment 
and in the fruit juice/brewing industry for the clarification and 
extraction of juices. 
In a preferred embodiment, the enzyme of the present invention is a 
thermostable enzyme which is stable to heat and is heat resistant and 
catalyzes the enzymatic hydrolysis of cellulose, i.e., the enzyme is able 
to renature and regain activity after a brief (i.e., 5 to 30 seconds), or 
longer period, for example, minutes or hours, exposure to temperatures of 
80.degree. C. to 105.degree. C. and has a temperature optimum above 
60.degree. C. 
The enzymes, their fragments or other derivatives, or analogs thereof, or 
cells expressing them can be used as an immunogen to produce antibodies 
thereto. These antibodies can be, for example, polyclonal or monoclonal 
antibodies. The present invention also includes chimeric, single chain, 
and humanized antibodies, as well as Fab fragments, or the product of an 
Fab expression library. Various procedures known in the art may be used 
for the production of such antibodies and fragments. 
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, 1975, Nature, 
256:495-497), the trioma technique, the human B-cell hybridoma technique 
(Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma 
technique to produce human monoclonal antibodies (Cole, et al., 1985, in 
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). 
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 the 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 "Methods for Measuring Cellulase 
Activities", Methods in Enzymology, Vol 160, pp. 87-116, which is hereby 
incorporated by reference in its entirety. Antibodies may also be employed 
as a probe to screen gene libraries generated from this or other organisms 
to identify this or cross reactive activities. 
The present invention is 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. 
In order to facilitate understanding of the following examples certain 
frequently occurring methods and/or terms will be described. 
"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, D. 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 may or may not have a 5' 
phosphate. Those that do not 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 the 
method of Sambrook, Fritsch and Maniatus, 1989. 
EXAMPLE 1 
Bacterial Expression and Purification of Endoglucanase 
An AEPII1a genomic library was constructed in the Lambda gt11 cloning 
vector (Stratagene Cloning Systems). The library was screened in Y1090 E. 
coli cells (Stratagene) for endoglucanase activity and a positive clone 
was identified and isolated. DNA of this clone was used as a template in a 
100 ul PCR reaction using the following primer sequences: 5' primer: 
AATAGCGGCCGCAAGCTTATCGACGGTTTCCATATGGGGATTGGTG (SEQ ID NO:3). 3' primer: 
AATAGCGGCCGCGGATCCAGACCAACTGG TAATGGTAGCGAC (SEQ ID NO:4). 
The PCR reaction product was purified and digested with Not I restriction 
enzyme. The digested product was subcloned into the pBluescript II SK 
cloning vector (Stratagene) and sequenced. The sequence information was 
used in the generation of primer sequences which were subsequently used to 
PCR amplify the target gene encoding the endoglucanase. The primer 
sequences used were as follows: 5' primer: 
TTTATTCAATTGATTAAAGAGGAGAAATTAACTATGATAAACGTTGC AACGGGAGAGGAG (SEQ ID 
NO:5) and 3' primer: TTTATTGGATCCTACTTTGTGTCAACGAAGTATCC (SEQ ID NO:6). 
The amplification product was digested with the restriction enzymes MfeI 
and BamHI. The digested product was then ligated to pQET cloning vector, a 
modified form of a pQE vector (Qiagen, Inc.) which was previously digested 
with BamHI and EcoRI compatible with MfeI. The pQE 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 amplified sequences were inserted in frame with the sequence encoding 
for the RBS. The ligation mixture was then used to transform the E. coli 
strain M15/pREP4 (Qiagen, Inc.) by electroporation. M15/pREP4 contains 
multiple copies of the plasmid pREP4, which expresses the lacI repressor 
and also confers kanamycin resistance (Kan.sup.r). Positive recombinant 
transformants were identified as having thermostable CMCase/endoglucanase 
activity by the assay described above. 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 lacI 
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. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings and, therefore, within the scope of the 
appended claims, the invention may be practiced otherwise than as 
particularly described. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 6 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1662 NUCLEOTIDES 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: DNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ATGATAAACGTTGCAACGGGAGAGGAGACCCCAATACACCTCTTTGGA48 
MetIleAsnValAlaThrGlyGluGluThrProIleHisLeuPheGly 
51015 
GTCAACTGGTTCGGCTTTGAGACACCGAACTACGTTGTTCACGGCCTA96 
ValAsnTrpPheGlyPheGluThrProAsnTyrValValHisGlyLeu 
202530 
TGGAGTAGGAACTGGGAGGACATGCTCCTCCAGATCAAGAGCCTTGGC144 
TrpSerArgAsnTrpGluAspMetLeuLeuGlnIleLysSerLeuGly 
354045 
TTCAATGCGATAAGGCTTCCCTTCTGTACCCAGTCAGTAAAACCGGGG192 
PheAsnAlaIleArgLeuProPheCysThrGlnSerValLysProGly 
505560 
ACGATGCCAACGGCGATTGACTACGCCAAGAACCCAGACCTCCAGGGT240 
ThrMetProThrAlaIleAspTyrAlaLysAsnProAspLeuGlnGly 
65707580 
CTTGACAGCGTCCAGATAATGGAGAAAATAATCAAGAAGGCTGGAGAC288 
LeuAspSerValGlnIleMetGluLysIleIleLysLysAlaGlyAsp 
859095 
CTGGGCATATTCGTGCTCCTCGACTACCACAGAATAGGATGCAACTTC336 
LeuGlyIlePheValLeuLeuAspTyrHisArgIleGlyCysAsnPhe 
100105110 
ATAGAACCCCTATGGTACACCGACAGCTTCTCGGAGCAGGACTACATA384 
IleGluProLeuTrpTyrThrAspSerPheSerGluGlnAspTyrIle 
115120125 
AACACCTGGGTTGAAGTCGCCCAGAGGTTCGGCAAGTACTGGAACGTT432 
AsnThrTrpValGluValAlaGlnArgPheGlyLysTyrTrpAsnVal 
130135140 
ATCGGCGCGGACCTGAAGAACGAACCCCACAGCTCAAGCCCCGCACCT480 
IleGlyAlaAspLeuLysAsnGluProHisSerSerSerProAlaPro 
145150155160 
GCCGCCTACACTGACGGAAGTGGGGCCACGTGGGGAATGGGCAACAAC528 
AlaAlaTyrThrAspGlySerGlyAlaThrTrpGlyMetGlyAsnAsn 
165170175 
GCCACCGACTGGAACCTGGCGGCTGAGAGGATAGGAAGGGCAATTCTG576 
AlaThrAspTrpAsnLeuAlaAlaGluArgIleGlyArgAlaIleLeu 
180185190 
GAGGTTGCCCCACAATGGGTTATATTTGTTGAGGGAACCCAGTTCACC624 
GluValAlaProGlnTrpValIlePheValGluGlyThrGlnPheThr 
195200205 
ACCCCCGAGATAGACGGTAGGTACAAGTGGGGCCACAACGCCTGGTGG672 
ThrProGluIleAspGlyArgTyrLysTrpGlyHisAsnAlaTrpTrp 
210215220 
GGCGGAAACCTTATGGGTGTTAGGAAGTACCCAGTTAACCTGCCCAGG720 
GlyGlyAsnLeuMetGlyValArgLysTyrProValAsnLeuProArg 
225230235240 
GACAAGGTTGTTTACAGCCCCCAAGTTTACGGTTCAGAAGTTTACGAC768 
AspLysValValTyrSerProGlnValTyrGlySerGluValTyrAsp 
245250255 
CAGCCCTACTTTGACCCCGGTGAGGGGTTCCCCGACAACCTCCCCGAA816 
GlnProTyrPheAspProGlyGluGlyPheProAspAsnLeuProGlu 
260265270 
ATATGGTACCACCACTTCGGCTACGTAAAGCTTGATCTCGGTTACCCT864 
IleTrpTyrHisHisPheGlyTyrValLysLeuAspLeuGlyTyrPro 
275280285 
GTTGTTATAGGTGAGTTCGGAGGCAAGTACGGCCATGGGGGAGACCCG912 
ValValIleGlyGluPheGlyGlyLysTyrGlyHisGlyGlyAspPro 
290295300 
AGGGATGTCACTTGGCAGAACAAGATAATAGACTGGATGATCCAGAAC960 
ArgAspValThrTrpGlnAsnLysIleIleAspTrpMetIleGlnAsn 
305310315320 
AAATTCTGTGACTTCTTCTACTGGAGCTGGAACCCAAACAGCGGTGAC1008 
LysPheCysAspPhePheTyrTrpSerTrpAsnProAsnSerGlyAsp 
325330335 
ACCGGTGGAATTCTGAAGGATGACTGGACGACAATATGGGAGGACAAG1056 
ThrGlyGlyIleLeuLysAspAspTrpThrThrIleTrpGluAspLys 
340345350 
TACAACAACCTGAAGAGGCTCATGGACAGCTGTTCTGGAAACGCCACT1104 
TyrAsnAsnLeuLysArgLeuMetAspSerCysSerGlyAsnAlaThr 
355360365 
GCCCCGTCCGTCCCCACGACAACTACAACAACAAGCACACCGCCAACG1152 
AlaProSerValProThrThrThrThrThrThrSerThrProProThr 
370375380 
ACCACAACGACTACAACATCCACTCCAACGACCACTACCCAGACCCCG1200 
ThrThrThrThrThrThrSerThrProThrThrThrThrGlnThrPro 
385390395400 
ACCACCACTACTCCAACTACGACAACCACCACGACCACAACTCCTTCA1248 
ThrThrThrThrProThrThrThrThrThrThrThrThrThrProSer 
405410415 
AATAACGTCCCATTTGAAATTGTGAACGTTCTCCCGACTAGCTCCCAG1296 
AsnAsnValProPheGluIleValAsnValLeuProThrSerSerGln 
420425430 
TACGAGGGAACCAGCGTGGAGGTTGTATGTGATGGAACCCAGTGTGCC1344 
TyrGluGlyThrSerValGluValValCysAspGlyThrGlnCysAla 
435440445 
TCCAGCGTTTGGGGAGCTCCGAACCTCTGGGGAGTCGTTAAAATCGGA1392 
SerSerValTrpGlyAlaProAsnLeuTrpGlyValValLysIleGly 
450455460 
AACGCCACCATGGACCCCAACGTTTGGGGCTGGGAGGACGTTTACAAG1440 
AsnAlaThrMetAspProAsnValTrpGlyTrpGluAspValTyrLys 
465470475480 
ACTGCACCCCAGGACATTGGAACCGGCAGCACAAAGATGGAGATAAGG1488 
ThrAlaProGlnAspIleGlyThrGlySerThrLysMetGluIleArg 
485490495 
AACGGGGTGCTCAAGGTTACAAACCTCTGGAACATCAACATGCATCCG1536 
AsnGlyValLeuLysValThrAsnLeuTrpAsnIleAsnMetHisPro 
500505510 
AAGTATAACACAATGGCATACCCGGAGGTCATATACGGCGCCAAGCCT1584 
LysTyrAsnThrMetAlaTyrProGluValIleTyrGlyAlaLysPro 
515520525 
TGGGGCAACCAGCCAATAAACGCTCCGAACTTCGTGCTCCCGATAAAG1632 
TrpGlyAsnGlnProIleAsnAlaProAsnPheValLeuProIleLys 
530535540 
GTCTCCCAGCTTCCGAGGATACTTCGTTGA1662 
ValSerGlnLeuProArgIleLeuArg 
545550 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 553 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(C) STRANDEDNESS: 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PROTEIN 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetIleAsnValAlaThrGlyGluGluThrProIleHisLeuPheGly 
51015 
ValAsnTrpPheGlyPheGluThrProAsnTyrValValHisGlyLeu 
202530 
TrpSerArgAsnTrpGluAspMetLeuLeuGlnIleLysSerLeuGly 
354045 
PheAsnAlaIleArgLeuProPheCysThrGlnSerValLysProGly 
505560 
ThrMetProThrAlaIleAspTyrAlaLysAsnProAspLeuGlnGly 
65707580 
LeuAspSerValGlnIleMetGluLysIleIleLysLysAlaGlyAsp 
859095 
LeuGlyIlePheValLeuLeuAspTyrHisArgIleGlyCysAsnPhe 
100105110 
IleGluProLeuTrpTyrThrAspSerPheSerGluGlnAspTyrIle 
115120125 
AsnThrTrpValGluValAlaGlnArgPheGlyLysTyrTrpAsnVal 
130135140 
IleGlyAlaAspLeuLysAsnGluProHisSerSerSerProAlaPro 
145150155160 
AlaAlaTyrThrAspGlySerGlyAlaThrTrpGlyMetGlyAsnAsn 
165170175 
AlaThrAspTrpAsnLeuAlaAlaGluArgIleGlyArgAlaIleLeu 
180185190 
GluValAlaProGlnTrpValIlePheValGluGlyThrGlnPheThr 
195200205 
ThrProGluIleAspGlyArgTyrLysTrpGlyHisAsnAlaTrpTrp 
210215220 
GlyGlyAsnLeuMetGlyValArgLysTyrProValAsnLeuProArg 
225230235240 
AspLysValValTyrSerProGlnValTyrGlySerGluValTyrAsp 
245250255 
GlnProTyrPheAspProGlyGluGlyPheProAspAsnLeuProGlu 
260265270 
IleTrpTyrHisHisPheGlyTyrValLysLeuAspLeuGlyTyrPro 
275280285 
ValValIleGlyGluPheGlyGlyLysTyrGlyHisGlyGlyAspPro 
290295300 
ArgAspValThrTrpGlnAsnLysIleIleAspTrpMetIleGlnAsn 
305310315320 
LysPheCysAspPhePheTyrTrpSerTrpAsnProAsnSerGlyAsp 
325330335 
ThrGlyGlyIleLeuLysAspAspTrpThrThrIleTrpGluAspLys 
340345350 
TyrAsnAsnLeuLysArgLeuMetAspSerCysSerGlyAsnAlaThr 
355360365 
AlaProSerValProThrThrThrThrThrThrSerThrProProThr 
370375380 
ThrThrThrThrThrThrSerThrProThrThrThrThrGlnThrPro 
385390395400 
ThrThrThrThrProThrThrThrThrThrThrThrThrThrProSer 
405410415 
AsnAsnValProPheGluIleValAsnValLeuProThrSerSerGln 
420425430 
TyrGluGlyThrSerValGluValValCysAspGlyThrGlnCysAla 
435440445 
SerSerValTrpGlyAlaProAsnLeuTrpGlyValValLysIleGly 
450455460 
AsnAlaThrMetAspProAsnValTrpGlyTrpGluAspValTyrLys 
465470475480 
ThrAlaProGlnAspIleGlyThrGlySerThrLysMetGluIleArg 
485490495 
AsnGlyValLeuLysValThrAsnLeuTrpAsnIleAsnMetHisPro 
500505510 
LysTyrAsnThrMetAlaTyrProGluValIleTyrGlyAlaLysPro 
515520525 
TrpGlyAsnGlnProIleAsnAlaProAsnPheValLeuProIleLys 
530535540 
ValSerGlnLeuProArgIleLeuArg 
545550 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 NUCLEOTIDES 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: Oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
AATAGCGGCCGCAAGCTTATCGACGGTTTCCATATGGGGATTGGTG46 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 42 NUCLEOTIDES 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: Oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AATAGCGGCCGCGGATCCAGACCAACTGGTAATGGTAGCGAC42 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 NUCLEOTIDES 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: Oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TTTATTCAATTGATTAAAGAGGAGAAATTAACTATGATAAACGTTGCAACGGGAGAGGAG60 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 35 NUCLEOTIDES 
(B) TYPE: NUCLEIC ACID 
(C) STRANDEDNESS: SINGLE 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: Oligonucleotide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TTTATTGGATCCTACTTTGTGTCAACGAAGTATCC35 
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