Note: Descriptions are shown in the official language in which they were submitted.
W094/00578 ~l 39 ~ 9 9 ~ PCT/AU93/00307
.
TITLE
"RECOMBINANT CELLULASES"
FIELD OF INVENTION
THIS INVENTION relates to recombinant
cellulases derived from anaerobic fungi and a method of
- production of recombinant cellulases and clones
utilised in the method.
BACKGROUND ART
Cellulose is one of the most abundant
polysaccharides in nature and consists of a polymer of
glucose linked by ~-1,4-glucosidic bonds. Conversion
of cellulose to simple sugars (cellobiose and glucose)
involves at least two types of hydrolases:
endoglucanases which hydrolyse internal ~-1,4-
glucosidic linkages in less ordered regions of
cellulose and exoglucanases tmainly cellobiohydrolases)
which cleave cellobiosyl units from non-reducing ends
of cellulose chains. Xylan, similar to the structure
of cellulose, consists of a backbone of ~-1,4-linked
xylose units. The enzymatic cleavage of ~-1,4-
xylosidic linkages is performed by endo-~-1,4-xylanases
(xylanases). These three types of enzymes usually
exist separately as individual proteins, each with
unique substrate specificity.
Many endoglucanases cleave only internal ~-1,
4-glucosidic linkages, producing rapid depolymerisation
of a model substrate, carboxymethyl cellulose (CM-
cellulose); whereas cellobiohydrolases are able to
hydrolyse crystalline cellulose and methylumbelliferyl
cellobioside tMUC) and have no or little depolymerising
activity against CM-cellulose. Similarly, many
xylanases exclusively attack ~-1,4-xylosidic linkages.
However, not all polysaccharide hydrolases have strict
substrate specificity. Due to the similarity in the
chemical nature of the substrates, cross specificity
occurs not only between two types of cellulase, but
also between cellulases and xylanases. A large number
~139~99 `
W094/00578 PCT/AU93/003~-
of cloned cellulases from bacteria have been reported
to possess some residual xylanolytic activity (usually
< 1%) or vice versa (Saarilahti et al., 1990; Yague et
al. 1990; Hazelwood et al., 1990; Flint et al., 1991;
Taylor et al., 1987).
Recent studies, based on partial enzyme
purification, showed that rumen anaerobic fungi such as
Neocallimastix frontalis might produce multi-functional
polysaccharide hydrolases (Gomez de Segura & Fevre,
1991; Li & Calza, 1991). Multi-functional poly-
saccharide hydrolases are of particular interest in
genetic manipulation of rumen bacteria to enrich for
the lignocellulose-degrading capacity. Simultaneous
enhancement of endoglucanase, cellobiohydrolase and
xylanase activities would facilitate the disruption of
the complex structure of lignocellulose, of which
cellulose and xylan are the major components. It may
also circumvent the rate-limiting problem which often
occurs when only one of a complex of enzymatic
reactions is enhanced.
Cellulose and hemicellulose (mainly xylan) are
major components of ruminants' diets, consisting of 50-
80% by weight of plant tissue. Effective utilisation
of plant feeds is therefore largely dependent on the
production of cellulolytic and xylanolytic enzymes by
microbial populations residing within the rumen.
Compared with other components of the diet, degradation
of cellulose and hemicellulose in the rumen is
relatively slow and incomplete; digestion may be as
low as 30% (Dehority, 1991). Thus, there is potential
economic value in enhancing the plant fibre-degrading
capacity by introducing plant polysaccharide hydrolase
gene(s) into rumen micro-organisms using recombinant
DNA techniques. The isolation of a gene encoding a
highly active enzyme able to degrade crystalline
cellulose in a ruminal environment is considered tQ be
one of the key steps in achieving this goal.
W094/00578 21 ~ o g ~ PCT/AU93/00307
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In the past decade, isolation of cellulase
genes from rumen micro-organisms was focused on
bacteria. Most of the cloned cellulases from the rumen
bacteria have little or no ability to degrade
crystalline cellulose (Robinson and Chambliss, 1989;
Hazlewood et al., 1990; Berger et al., 1989; Romaniec
et al., 1989; Flint et al., 1989), though a few cloned
cellulases exhibit some significant activity towards
this substrate (Cavicchioli and Watson, 1991; Howard
and White, 1988).
The anaerobic fungus Neocal l imastix
patriciarum, isolated from the sheep rumen, has a high
capacity for cellulose degradation and can grow on
cellulose as the sole carbohydrate source (Orpin &
Munn, 1986; Williams & Orpin, 1987).
Molecular biological aspects of fungal
cellulases have been studied mainly in the aerobic
fungi (Shoemaker et al., 1983; Teeri et al., 1983;
Chen et al., 1987; Sims et al., 1g88; Azevedo et al.,
1990). These studies have rapidly elucidated the
complexity, structure and regulation of aerobic fungal
cellulases. However, molecular characterisation of
anaerobic fungal cellulases has been hampered by lack
of information on the successful purification of
individual cellulolytic enzymes from the fungal
cellulase complexes. Thus, the preparation of
- antibodies or protein microsequencing for the design of
oligonucleotide probes has not been possible.
Reference may also be made to other prior art
which serves as background prior art prior to the
advent of the present invention. Such prior art
includes:
(i) Reymond et. al. FEMS Microbiology letters
(1991) 107-112;
(ii) Orpin et. al. Current Microbiology Vol 3
(1979) pp 121-124;
WO 94/00578 ~ 1 3 q ~ pcr/Au93/oo3o~
(iii) Mountfort and Asher in "The Roles of Protozoa
and Fungi in Ruminant Digestion" (1989)
Pernambul Books (Australia);
(iv) Joblin et. al. FEMS Microbiology Letters 65
(1989) 119--122;
(v) Lowe et. al. Applied and Environmental
Microbiology June 1987 pp 1210-1215; and
(vi) Lowe et. al. Applied and Environmental
Microbiology June 1987 pp 1216-1223.
Cloning of cellulase genes from bacteria can
be achieved by isolation of enzymatically active clones
from genomic libraries established in E. col i . However
this approach for isolation of cellulase genes from
fungal genomic libraries with functional expression of
15 cellulase is usually not possible. This is because
fungi are eucaryotic microorganisms. Most eucaryotic
genes contain introns and E. col i is unable to perform
post-transcriptional modification of mRNAs in order to
splice out introns. Therefore, enzymatically
20 functional protein cannot normally be synthesised in
clones obtained from a fungal genomic library.
The cDNA cloning approach can be used to
overcome the post-transcriptional modification problem
in E. col i . However, cellulases in fungi are usually
25 glycosylated and glycosylation is often required for
biological activity of many glycosylated enzymes. E.
col i lacks a glycosylation mechanism. This problem can
be solved if the cloned gene is transferred to an
eucaryotic organism, such as yeast. Other problems
30 which are often encountered in obtaining a biologically
functional protein from a cDNA clone in E. col i are (i)
that many eucaryotic mRNAs contain translational stop
codons upstream of the translational start codon of a
gene which prevents the synthesis of the cloned protein
35 from the translational start provided in the vector,
and (ii) that synthesis of the cloned protein is based
on fusion proteins and the biological function of the
W094/00578 ~ 1 3 9 a 9 9 PCT/AU93/00307
cloned protein is often adversely affected by the fused
peptide derived from the cloning vector.
Therefore, in the past, researchers in this
field employed differential or cross hybridisation,
antibody probes or oligonucleotide probes for the
isolation of fungal polysaccharide hydrolase cDNA or
genomic DNA clones. Relevant publications in this
regard include Reymond et. al.; Teeri et. al., referred
to above; Shoemaker et. al. referred to above; Sims
et. al. referred to above; Morosoli and Durand FEMS
Microbiology Letters 51 217-224 (1988); and Azevedo
et. al. referred to above. However, these methods are
very time-consuming, and quite often two stages of
intensive cloning work are required for isolation of an
enzymatically functional clone. For antibody or
oligonucleotide probes, purification of the fungal
cellulase is also required. It usually takes more than
one year to obtain a functional enzyme clone using the
above approaches.
Isolation of fungal cellulase cDNAs by
utilising an expression system in E. coli, has not been
reported prior to the advent of this invention,
probably at least partially due to failure in obtaining
enzymatically functional cellulase clones resulting
from the use of inappropriate expression vectors.
Selection of expression vector systems is important.
If plasmid expression vectors such as pUC vectors are
used, and the cloned enzyme is trapped inside the cell,
screening for cellulase clones by the convenient
cellulose-agar plate technique becomes difficult.
Bacteriophage vectors have an advantage in respect to
the release of the cloned enzyme into cellulose-agar
medium due to cell lysis. However, commonly used
bacteriophage expression vectors, ~gt11 and its
derivatives, have polyclonal sites at the C-terminus of
the LacZ peptide. The large part of LacZ peptide fused
to the cloned enzyme often adversely affects the cloned
W094/00578 2134 ~ ~ 9 PCT/AU93/0030-
enzyme activity.
In specific regard to the abovementioned
Reymond et. al. (1991 ) reference there is described an
attempt of molecular cloning of polysaccharide
hydrolase (ie. cellulase) genes from an anaerobic
fungus which is N. frontalis. In this reference a
clone from a cDNA library derived from N. frontalis
hybridized to a DNA probe encoding part of the exo-
cellobiohydrolase (CBH 1) gene of Trichoderma reesei.
However it was subsequently revealed by Reymond et. al.
in a personal communication that the particular cDNA
clone obtained from N. frontalis does not encode any
polysaccharide hydrolase.
Moreover the Reymond et. al. reference did not
describe the production of biologically functional
enzymes from these clones.
BROAD STATEMENT OF INVENTION
It is an object of the invention to provide a
recombinant cellulase from an anaerobic rumen fungus
which may be of use commercially in relation to
hydrolysis of cellulose or cellulose derivatives
including plant cell walls.
A further object of the invention is to
provide a method of cloning of cellulase cDNAs from an
anaerobic rumen fungus which may encode the recombinant
cellulase of the invention.
A further object of the invention is to
provide cellulase clones which may be produced in the
abovementioned method.
The method of cloning of the invention
includes the following steps:
(i) cultivation of an anaerobic rumen
fungus;
(ii) isolating total RNA from the culture
in step (i);
(iii) isolating poly A~ mRNA from the total
RNA referred to in step (ii);
W O 94/00578 PC~r/A U93/00307
213'qOg9
(iv) constructing a cDNA expression
library;
(v) ligating cDNAs to a bacteriophage
~ expression vector selected from ~ZAP,
~ZAP II or vectors of similar
properties;
(vi) screening of cellulase positive
recombinant clones in a culture medium
incorporating cellulose by detection
of cellulose hydrolysis; and
(vii) purifying cellulase positive
recombinant clones.
In step (i) above in relation to preparation
of the recombinant cellulase, from anaerobic fungi,
particularly alimentary tract fungi, may be cultivated
as described hereinbelow. These fungi are strict
anaerobes and may be exemplified by Neocallimastix
patriciarum, Neocallimastix frontalis, Neocallimastix
hurleyensis, Neocallimastix stanthorpensis,
Sphaeromonas communis, Caecomyces equi, Piromyces
communis, Piromyces equi, Piromyces dumbonica,
Piromyces lethargicus, Piromyces mai, Ruminomyces
elegans, Anaeromyces mucronatus, Orpinomyces bovis and
Orpinomyces joyonii. In regard to the above mentioned
anaerobic alimentary tract fungi, Caecomyces equi,
Piromyces equi, Piromyces dumbonica and Piromyces mai
are found in horses and thus are not located in the
rumen of cattle like the other fungi described above.
The cultivation may proceed in appropriate
culture media containing rumen fluid and also may
contain cellulose such as Avicel (ie. a form of
microcrystalline cellulose) as a carbon source under
anaerobic conditions. After cultivation of the fungi
total RNA may be obtained in any suitable manner. Thus
initially the fungal cells may be harvested by
filtration and subsequently lysed in appropriate ,cell
lysis buffer by mechanical disruption. A suitable RNA
W094/00578 % 1 3q Q 9 9 PCT/AU93/003~-
preserving compound may also be added to the fungal
cells to maintain the RNA intact by denaturing RNAses
which would otherwise attack the fungal RNA. The total
RNA may subsequently be isolated from the homogenate by
any suitable technique such as by ultracentrifugation
through a CsCl2 cushion or alternative technique as
described by Sambrook et. al. in Molecular Cloning; A
Laboratory Manual 2nd Edition Cold Spring Harbor
Laboratory Press in 1989. An alternative method for
preparation of total fungal RNA to that described above
may be based on or adapted from the procedure described
in Puissant and Houdebine in Bio-Techniques 148-149 in
1990 or by the method of Chomczynski and Sacchi in
1987. Total fungal RNA in this alternative technique
may also be isolated from the above homogenate by
extraction with phenol chloroform at pH4 to remove DNA
and associated protein. Total RNA obtained was further
purified by washing with lithium chloride-urea
solution.
Poly (A)+ mRNA may then be isolated from the
total RNA by affinity chromatography on a compound
containing multiple thymine residues such as oligo (dT)
cellulose. Alternatively a compound containing
multiple uracil residues may be used such as poly (U)-
Sephadex. The poly (A) t mRNA may then be eluted from
the affinity column by a suitable buffer.
A cDNA expression library may then be
constructed using a standard technique based on
conversion of the poly (A) t mRNA to cDNA by the enzyme
reverse transcriptase. The first strand of cDNA may be
synthesised using reverse transcriptase and the second
strand of the cDNA may be synthesised using E. coli DNA
polymerase I. The cDNA may subsequently be fractionated
to a suitable size and may be ligated to the
bacteriophage expression vector, preferably ~ZAP or
~ZAPII. The cDNA library may then be amplified after
packaging in vitro, using any suitable host bacterial
W094/00578 ~1 3 q o ~ 9 PCT/AU93/00307
._
cell such as a suitable strain of E. col i .
The choice of the bacteriophage expression
vector in step (v) is important in that such expression
~ vector should include the following features:
(i) having an E. coli promoter;
(ii) having a translation start codon;
(iii) having a ribosomal binding site;
(iv) the fusion peptide derived from the
vector should be as small as possible,
as the biological function of the
cloned protein is usually adversely
affected by the fused peptide derived
from the vector. Therefore the
polyclonal sites of the bacteriophage
expression vector are suitably located
at the N-terminus of lacZ peptides
such as in AZAPII.
It will be appreciated from the foregoing that
if an expression vector is utilised as described above
the chances of obtaining a biologically functional
enzyme is greatly increased. Isolation of many
enzymatically functional cellulase clones in the
present invention as described hereinafter has proved
the efficiency of this approach. To our knowledge this
is the first record of isolation of cellulase cDNA
clones with functional enzyme activity from anaerobic
fungi based upon the expression of recombinant
bacteriophage in E col i using an expression vector such
as that described above. ~ZAP and ~ZAP II are examples
of such expression vectors.
Therefore the term "vectors of similar
properties" to ~ZAP or ~ZAPII includes within its scope
expression vectors having the abovementioned features
(i) (ii), (iii) and (iv).
It is also clear from the product summary
which accompanies the ~ZAPII vector as supplied by the
manufacturer that in relation to fusion protein
W094/00578 2 ~ ~q 9 g ` PCT/AU93/0030~
1 0
expression that such fusion proteins may only be
screened with antibody probes. Clearly there was no
contemplation that the ~ZAPII vector could be utilised
for screening of clones involving enzymic activity on a
suitable substrate or any direct screening by
biological activity. When it is realised that the
present invention involves expression in a bacterial
host cell such as E col i of a cDNA of eucaryotic origin
(ie. fungal origin) then the novelty of the present
invention is emphasised.
The screening of cellulase positive
recombinant clones may be carried out by any suitable
technique based on hydrolysis of cellulose. In this
procedure the clones may be grown on culture media
incorporating cellulose and hydrolysis may be detected
by the presence of cellulase-positive plaques suitably
assisted by a suitable colour indicator. Cellulase
positive recombinant clones may then be purified and
the cDNA insert in the clones may then be excised into
pBluescript (SK(-)).
Any suitable E. col i promoter may be used in
the expression vector described above. Suitable
promoters include lacZ, Tac, Bacteriophage T7 and
lambda-PL -
The recombinant cellulases may then be
characterised and principal features that have been
ascertained are as follows:
(i) The cloned celA enzyme has high specific
activity on crystalline and amorphous
cellulase. The optimal pH and temperature for
cellulose hydrolysis are pH5 and 40C,
respectively.
(ii) The cloned celD enzyme is a multi-functional
cellulase with a high activity of
endoglucanase, cellobiohydrolase and xylanase.
The optimal pH and temperature for cellulose
hydrolysis are at pH5 and 40C, respectively.
W094/00578 %13~ ogg PCT/AU93/00307
.. _
1 1
(iii) celD cDNA can be truncated to code for three
catalytically active domains. Each domain has
endoglucanase, cellobiohydrolase and xylanase
activity and cellulose-binding capacity.
(iv) The recombinant celA and celD enzymes also
have very high activity on lichenan.
(v) A combination of celA and celD enzymes can
hydrolyse crystalline cellulose more
efficiently.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Method
Microbial strains, vectors and culture media.
The anaerobic fungus Neocal l imastix
patriciarum ( type species) was isolated from a sheep
rumen by Orpin & Munn (1986) and cultivated in the
laboratory for many years under selection by
lignocellulose substrate. The culture medium for N.
patriciarum was described previously (Kemp et al.,
1984). Microcrystalline cellulose (Avicel) was used as
the sole carbohydrate source. Host strains for cDNA
cloning were E col i PLK-F and XL1-Blue obtained from
Stratagene. E col i strains were grown in L-broth
(Sambrook et al., 1989). ~ZAPII vector was obtained
from Stratagene and the recombinant phage were grown in
E coli strains according to the supplier~s
instructions.
RNA isolation.
Frozen fungal mycelia were ground into fine
powder with a mortar and pestle under liquid N2.
Powdered mycelia were homogenised in guanidinium
thiocyanate solution (4M guanidinium thiocyanate, 0.5%
(w/v) sodium lauryl sarcosine, 25 mM-sodium citrate,
pH7.0, 1mM-EDTA and 0.1 M-~-mercaptoethanol) using a
mortar and pestle for 5 min and ther. further
homogenised with a Polytron at full speed for 2 min.
Total cellular RNA was prepared from the homogenate
either by ultracentrifugation through a CsCl2 cushion
W094/00578 PCT/AU93/003
2 13 q 09 g 12
(Sambrook et al., 1989) or by the method of Chomczynski
& Sacchi (1987) with the following modifications. The
RNA pellet, obtained after acid guanidinium
thiocyanate/phenol/chloroform extraction and the first
step of 2-propanol precipitation, was suspended in a
LiCl/urea solution (6 M-urea, 3 M-LiCl, 1 mM-EDTA, pH
7.6). The suspension was shaken at 4C for 1-2 h to
remove contaminating protein and DNA. After
centrifugation, the RNA pellet was briefly washed once
with the LiCl/urea solution, twice with 75% (v/v)
ethanol and then dissolved in 10 mM-Tris/HC1/1 mM-EDTA,
pH 8Ø The RNA was further purified by extraction
with phenol/chloroform and ethanpol precipitation.
Poly(A)+ RNA was selected by oligo(dT)-cellulose
chromatography (Sambrook et al. 1989).
General recombinant DNA techniaues.
DNA isolation, restriction endonuclease
digestion, ligation, transformation and preparation of
RNA probes were performed basically according to
procedures described by Sambrook et al. (1989).
Construction and screeninq of the N. Patriciarum cDNA
library.
Double-stranded cDNA was synthesised from mRNA
isolated from N. patriciarum grown on the medium
containing 1% (w/v) Avicel for 48 h and ligated with
~ZAPII using a ZAP-cDNA synthesis kit, according to the
manufacturer's instructions (Stratagene). A cDNA
library of 106 recombinants was obtained. Recombinant
phage were screened for cellulolytic activity by
plating in 0.7% (w/v) soft agar overlays containing one
of the following substrates 0.5% (w/v)
carboxymethylcellulose (CM-cellulose), 1 mm MUC or 0.1%
xylan. 10 mM-isopropyl ~-D-thiogalactopyranoside
(IPTG; an inducer for lacZp-controlled gene expression)
was also included. CM-cellulose hydrolysis was
detected by the Congo red staining procedure (Teather &
Wood, 1982). MUC hydrolysis was examined for
W094/00578 PCT/AU93/00307
~- 213~9
13
fluorescence under UV light. The cDNA inserts in CM-
cellulose positive phage were recovered in the form of
pBluescript (SK-) by in vivo excision, according to
~ Stratagene~s instructions.
Construction of deletion mutants.
Deletion of celD cDNA was achieved by either
removing a cDNA fragment with restriction enzymes or by
exonuclease III digestion (Sambrook et al., 1989). The
truncated celD cDNA was checked either by restriction
mapping or by partial nucleotide sequencing at the
insert terminals.
DNA sequencinq.
Single-stranded plasmid DNA was prepared
basically according to Stratagene's protocol.
Sequencing of the resultant DNA was performed using
dideoxynucleotide method (Tabor and Richardson, 1987).
Southern blot hYbridisation.
~ DNA from the cellulase-positive clones was
purified by 2 rapid mini-preparation method as follows.
One millilitre of phage lysate from liquid culture was
incubated with RNAase A (10~g ml~') and DNasel (1~g ml~l)
at 37C for 1 h and with proteinase K (1 mg ml~l) at
37C for 3 h and then extracted with phenol/chloroform.
The DNA was precipitated by ethanol, digested with
EcoR1 and Xhol (the cDNA cloning sites), fractionated
by electrophoresis on 1% (w/v) agarose gel and blotted
onto Hybond N membrane (Amersham). Procedures for
hybridisation and signal detection were as described
previously (Xue & Morris, 1992), using digoxigenin-
labelled RNA probes prepared from the 3'-region-deleted
cDNA. Hybridisation was carried out at 50C in a
hybridisation mixture of 50% (v/v) formamide, 0.8 M-
NaCl, 50 mM-sodium phosphate (pH 7.2), 4mM-EDTA, 0.2%
(w/v) SDS. 5x Denhardt's solution, 0.2 mg yeast RNA
ml~l , 0.2 mg herring sperm DNA ml~l (1 x Denhardt~s
solution is 0.02% bovine serum albumin, 0.02% Ficoll,
0.02% polyvinylpyrrolidone). High-stringency washing
21~q ~99
W094/00578 ~ PCT/AU93/0030'
14
was performed in 0.1 x SSC/0.1% (w/v) SDS at 68C (1 X
SSC is 0.15 M-NaCl, 15 mM-sodium citrate).
EnzYme assaYs, cellulose-bindinq studies and Product
identification.
E coli cells harbouring the recombinant
plasmids were grown in LB medium to the end of the
exponential phase in the presence of 1mM IPTG. Crude
cell lysates prepared according to Schwarz et al.
(1987) were used as enzyme sources. For standard
quantitive assays, the enzyme preparations were
incubated at 39C for 30-60 min in 50 mM-sodium citrate
(pH 5.7) with the following substrates: 0.5% (w/v) CM-
cellulose (low viscosity, Sigma, 1% (w/v) amorphous
cellulose (H3PO4-swollen Avicel), 1% (w/v) Avicel
(Merck), 0.05% (w/v) p-nitrophenyl cellobioside (pNPC,
Sigma), p-nitrophenyl glucopyranoside (pNPG, Sigma),
0.25% (w/v) oat spelt xylan (Sigma) and 0.4% Lichenan.
The reducing sugars released from cellulose, Lichenan
or xylan were measured as described by Lever (1972).
The p-nitrophenyl groups released from p-nitrophenyl
derivatives were measured as described by Deshpande et
al. (1988). The cell lysate prepared from E coli
strain XL1-Blue harbouring non-recombinant pBluescript
was used as control. Protein concentrations were
determined by dye-bindin5 assay using the Bio-Rad
protein assay kit II according to the supplier~s
instructions. Qualitative assays were performed using
0.8% (w/v) agarose gel plates containing 0.2% (w/v) CM-
cellulose, lichenan, laminarin or xylan or 1 mM MUC in
50 mM Na-citrate pH5.7. Hydrolysis zones were detected
as described above.
For assays of cellulose-binding capacity of
the cloned cellulase, cell lysates were incubated with
200~1 of pre-washed 5% (w/v) Avicel in 50 mM-sodium
citrate (pH 5.7) at 0C with continuous shaking for 1h.
The unbound protein was removed after centrifugation
and the Avicel pellet was washed three times with 50mM-
W094/00578 2~ ~q O PCT/AU93/00307
sodium citrate (pH 5.7). The bound cellulase was
assayed for enzyme activity as above.
For analysis of hydrolysis products of
~ cellulosic substrates, crude E coli lysates containing
the cloned cellulases were spin-dialysed to remove
small molecules using Centricon concentrators (Amicon).
The dialysed enzyme preparations were incubated at 39C
in 50 mM-sodium citrate (pH 5.7) with 1% (w/v) Avicel
or cellodextrins (2 mg ml~l) containing 3-6 glucose
units. In order to examine the intermediate and end
hydrolysis products of cellodextrins, samples were
taken at five incubation times (30 min, 1 h, 2h, 4h
and 30h), using appropriate amounts of enzymes to
ensure partial as well as complete digestion.
Hydrolysis products of cellulosic substrates were
identified by thin-layer chromatography (TLC) using
silica gel plates and a solvent system of ethyl
acetate/water/methanol (3:3:4, by vol.). The positions
of sugars on the plate were visualised by spraying with
the diphenylamine reagent as described by Lake &
Goodwin (1976) and authentic cellodextrins (Merck) were
used for identification.
Results and Discussion
Isolation of cellulase cDNAs from N. Patriciarum cDNA
exPression librarY.
A cDNA library was prepared from poly (A)+ RNA
isolated from N. patriciarum grown on Avicel as the
sole carbohydrate source and was constructed in E coli
using a AZAPII vector. The library was initially
screened for expression of endoglucanase activity on
CM-cellulose plates. Two hundred CM-cellulose positive
plaques were identified after screening 4 x 105
plaques from library. These CM-cellulose positive
clones were screened for cellobiohydrolase activity
first on MUC plates and were further tested for the
ability to hydrolyse microcrystalline cellulose by
assaying the reducing sugar released after absorption
W094/00578 , PCT/AU93/0030'
213~ O~g ~
` 16
of cellulase in the supernatant of the recombinant
bacteriophage lysates to Avicel followed by incubation
at 39C for 3 hr (see cellulose-binding assay in
Method). Eleven bacteriophage clones exhibited large
hydrolysis zones on both CM-cellulose and MUC plates,
as well as activity towards Avicel. These eleven
clones were then tested for xylanolytic activity on
xylan plates and all were positive.
Analysis of the selected clones by restriction
mapping revealed that ten of the eleven clones (the
size of cDNA inserts ranging from 1.6 Kb to 3.9 Kb)
shared the same restriction pattern. A restriction map
of the longest cellulase cDNA sequence, designated celD
(pCNP4.1) is shown in Fig. 1. The remaining clone
possessed an insert of 7.0 Kb designated as celE and
also had a similar restriction pattern to celD, but
contained two additional 1.15-Kb internal EcoR1-EcoR1
fragments and a 1.7 Kbp cDNA (Fig 1). Cross
hybridisation analysis showed that CelD strongly
hybridised to CelE using a nucleic acid probe prepared
from CelD cDNA in which the 3' region was deleted.
Thus it is most likely that the ten clones originate
from the same gene and the celE clone is a related cDNA
to celD.
Three other classes of cellulase cDNAs were
isolated from the pool of CM-cellulose-positive clones
by restriction mapping and cross-hybridisation.
Restriction maps of three cellulase cDNAs (the longest
cDNA insert for each type), designated celA ( 2.0 Kb),
CelB (1. 7 Kb) and CelC (1. 6 Kb) respectively, are shown
in Fig. 2. Southern hybridisation analysis showed
these three cDNA inserts did not cross-hybridise to
each other (Fig. 3), using nucleic acid probes prepared
from CelA and CelC clones with the 3~regions of the
cDNA insert removed by digestion with Xhol and the
enzyme at the upstream restriction site (see Fig 2).
Similarly, CelD did not hybridise to celA, celB and
W094/00578 ~ 1 3 q O 9 9 PCT/AU93/00307
._ .
1 7
CelC using high stringency conditions.
Enzymatic ProPerties of the Recombinant cellulases
The substrate specificity of these recombinant
~ cellulases was further characterised by quantitative
measurement of the activity on various cellulosic
substrates and xylan. As shown in Table 1, the celD
enzyme was most active on CM-cellulose, but it also
possessed cellobiohydrolase-like properties, as it was
highly active on crystalline cellulose, MUC and p-
nitrophenyl cellobioside (pNPC) as well as amorphous
cellulose. The enzyme showed no activity on
methylumbelliferyl glucoside (MUG) and p-nitrophenyl
glucoside (pNPG), substrates for ~-glucosidase. Other
cellulosic substrates tested were lichenan (a mixed
glucan containing ~-1,4 and ~-1,3 linkages) and
laminarin (predominantly ~-1,3-glucan). The celD
enzyme had very high activity towards lichenan (Table
1) and produced a large hydrolysis zone on lichenan-
containing agarose gel plates, but did not produce a
hydrolysis zone on laminarin plates (Fig. 4). This
indicates that cleavage on lichenan is at the ~-1,4-
linkages. Interestingly, a high xylanase activity was
also present in the celD enzyme. Analysis of
hydrolysis products by TLC showed that the celD enzyme
was able to hydrolyse cellodextrins (containing 3-S
glucose units) to glucose and cellobiose. Its
catalytic mode on these cellulosic substrates is of a
typical endoglucanase (ie. it cleaved ~-1,4-glucosidic
linkages at random positions, as shown in Fig. 5).
However, the hydrolysis products of microcrystalline
cellulose were mainly cellobiose with a trace amount of
glucose (Fig.5), indicative of cellobiohydrolase
activity. It appears that it is a truly multi-
functional plant polysaccharide-degrading enzyme.
Although a number of cellulases and xylanases have been
shown to have multiple substrate specificity, most of
them possess only residual activity (usually < 1%)
W094/00578 ~ l ~q 0 9 ~ PCT/AU93/003
18
towards the secondary substrate (Saarilahti et al.,
1990; Yague et al., 1990; Hazlewood et al., 1990;
Flint et al., 1991; Taylor et al., 1987). The
substrate specificity of celE enzyme is similar to celD
enzyme, but its activity was about 4-fold lower. The
enzyme encoded by celA possesses cellobiohydrolase
properties. It has very high activity in hydrolysis of
crystalline and amorphous cellulose, although it also
has relatively weak activity on CM-cellulose (Table 1).
The cellobiohydrolase-like properties of the celA
enzyme was further confirmed by its hydrolysis pattern
as cellobiose was the only product released from
cellotetrose or Avicel by the celA enzyme (Fig. 6).
The celA enzyme also has very high activity on Lichenan
and no activity on laminarin. The enzyme properties of
celB and celC resembled endo-glucanase (Table 1 and
Fig. 6).
The pH and temperature profiles of celA and
celD enzymes are shown in Fig. 7 and Fig. 8. The celA
and celD enzymes were active from pH4.5 to pH8.5 and
preferably at pH5-7. The thermostability of these
enzymes was tested at temperature from 30C-60C. The
celA and celD enzymes are active preferably at 30C-
50C. The recombinant enzymes remain active in
hydrolysis of Avicel at 39C for at least 21 hr (Fig. 9
and Fig. 10). The hydrolysis rates of Avicel by celA
or celD enzyme were not proportional to the enzyme
levels tested (Fig. 9 and Fig. 10). However, a
combination of celA and celD enzymes performs much
better in hydrolysis of Avicel than doubling the
concentration of individual enzyme (Fig. 11),
suggesting a complementary effect of the celA and celD
enzymes.
It has also been ascertained that recombinant
xylanases may be produced by the method of the
invention using substantially the same experimental
protocols described above. One such xylanase termed
W094/00578 ~1 3 4 0 9 g ~ i PCT/AU93/00307
.~
1 9
pNX-Tac is a DNA construct as shown in Fig. 16 and has
a DNA sequence as shown in FIG 17.
A combination of a recombinant xylanase such
~ as pNX-tAC and celA and celD enzyme has demonstrated
that co-operativity or synergy may occur in relation to
biological activity on crude cellulosic substrates
containing lignin and hemicellulose components. This
activity is shown in Table 2.
Cellulose-bindinq caPacitY of celA and celD enzymes
The cellulose-binding capacity of the celA and
celD enzymes were assessed by a comparative assay of
the enzyme activity with or without prior absorption to
crystalline cellulose (Avicel). The amount of reducing
sugar released from Avicel after absorption of the
enzyme to Avicel followed by extensive washing of the
enzyme-substrate complex was 23.3 ~g glucose equivalent
min I per mg protein (the crude cell lysate
preparation), compared to 24.3 ~g min I per mg protein
for the enzyme added without prior absGrption. This
high recovery (95%) of the enzyme activity after
absorption and washing suggests that the celD enzyme
possesses a strong cellulose-binding capacity. The
recovery of celA enzyme after adsorption to Avicel and
washing was 77%, slightly lower than celD enzyme.
Presumably, the cellulose-binding capacity is important
for efficient degradation of cellulose as a result of
the close contact of the enzyme with this insoluble
substrate.
Functional domains of celD enzYme
To investigate the locations of catalytic and
cellulose-binding domains of the celD enzyme and to
elucidate whether the multiple substrate specificity of
the enzyme is due to the presence of different
catalytic domains, a series of deletion analyses of
celD cDNA was conducted. As shown in Fig. 12, celD
cDNA can be truncated to code for three catalytically
active domains, when each domain was fused in frame
WO 94/00578 ~ l 3 q ~ 9 9 ~ ,~ , PCI/AU93/00307
with the vector's lacZ translation initiation codon.
These are designated domain I (pCNP4.2), domain II
(pCNP4.4) and domain III (pCNP4.8), respectively. The
subclone construction of domain I was obtained by
deletion of a 2.75-Kb fragment at the 3~region of celD
cDNA (the PvulII--Xhol fragment). Domain II contained
sequence from the position 1.15 Kb to 2.3 Kb of celD
cDNA and domain III from 2.3 Kb to 3.37 Kb. The
subclone construction of domain II (pCNP4.4) was
achieved by deletion of a 1.15-Kbp EcoRI-PvuII fragment
at the 5' region and exonuclease III digestion at the
3I region of celD cDNA and domain III by exonuclease
III digestion from both the 5' region and 3I region of
the celD. Interestingly, all three domains possessed
the same pattern of substrate specificities as the
enzyme produced by the untruncated celD cDNA.
Moreover, all three domains had cellulose-binding
capacity. Recovery of the enzyme activity after
absorption to Avicel and subsequent washing ranged from
70% to 80%. This is slightly lower than the enzyme
from the untruncated celD cDNA.
The celD cDNA was sequenced (see Fig. 13) and
graphical presentation of celD structure is shown in
Fig. 14. The amino acid sequences of three catalytic
domains deduced from the nucleotide sequence are
presented in Fig. 15. In the untruncated celD, the
third catalytic domain is untranslated, because there
is a translation stop codon at the end of the second
domain.
Overall functional analysis has revealed the
novel properties of celD enzyme. Although some
cellulases and xylanases consist of two mono-functional
catalytic domains (Saul et al., 1990; Gilbert et al.
1992) or possess a single multi-functional domain
(Foong et al., 1991), there is no previous example of a
polysaccharide hydrolase cDNA encoding three
multifunctional catalytic domains, with each catalytic
W094/00578 ~1 ~q ~ PCT/AU93/00307
. _
21
domain possessing cellulose-binding capacity. A multi-
functional enzyme would be beneficial for the rumen
fungus in its natural environment where these
~ polysaccharide substrates exist in a complex structure.
Usually, several types of polysaccharide hydrolases are
required to form a multi-enzyme complex acting co-
operatively on these natural substrates.
Main features of celD cDNAs from the rumen anaerobic
funqus, Neocallimastix Patriciarum
1. celA cDNA encodes a highly active
cellobiohydrolase which efficiently hydrolyses
both crystalline and amorphous cellulose.
2. celD cDNA encodes a highly active enzyme with
endoglucanase, cellobiohydrolase and xylanase
activities, capable of degrading a wide range
of cellulosic materials and xylan.
3. The cloned celD enzyme can actively hydrolyse
crystalline cellulose, presumably due to the
presence of both endoglucanase and
cellobiohydrolase activities which act
synergistically in cellulolysis.
4. The celD cDNA contains sequences which can
encode three functional domains; each domain
possesses endoglucanase, cellobiohydrolase and
xylanase activities in addition to strong
cellulose-binding capacity. The cellulose-
binding capacity is important for efficient
degradation of cellulose as a result of the
close contact of the enzyme with this
insoluble substrate.
5. celA and celD enzymes have very high activity
in hydrolysis of lichenan.
A multi-functional enzyme could more
efficiently degrade the polysaccharide complex existing
in plant materials. Although a number of cloned
cellulases showed multiple substrate specificity, most
of them possess only residual activity (usually < 1%)
W094/00578 2 ~ 3 q O 9 g ~ PCT/AU93/0030'
22
towards the secondary substrate. There is no previous
example of a cellulase or xylanase gene encoding three
multi-functional catalytic domains with each possessing
strong cellulose-binding capacity. The activity of the
cloned celA and celD enzymes in E col i can be further
increased by using stronger promoters.
Potential aPPlications of celA and celD cDNAs
Cellulose and hemicellulose (consisting mainly
of xylan) represent the most abundant natural resource
on earth. Cellulose alone accounts for about 40% total
biomass with an annual production of 4 x 10l tons
(Coughlan, 1985), which was equivalent to 70 kg of
cellulose synthesises per person each day, as
calculated in 1983 by Lutzen et al. (1983). Most plant
materials consist of 40-60% cellulose and 15-30~
hemicellulose (Dekker and Lindner 1979). Efficient
utilisation of plant materials by ruminant animals,
such as sheep and cattle, are therefore largely
dependent on production of cellulolytic and xylanolytic
enzymes by microbial populations residing within the
rumen (the enlarged forestomach of the ruminants).
Compared with other components of the diet, degradation
of cellulose and hemicellulose in the rumen is
relatively slow and it can be as low as 30% (Dehority,
1991). Thus, there is potential economic value in
enhancing the plant fibre-degrading capacity of rumen
micro-organisms by introducing plant polysaccharide
hydrolase gene(s) using recombinant DNA techniques.
Isolation of a gene encoding a highly active enzyme
which is able to degrade crystalline cellulose and
xylan in a ruminal environment is considered to be one
of the key steps in achieving this goal. celA and celD
cDNAs, isolated from a rumen anaerobic fungus, may
possess advantages over other cellulase genes from non-
ruminal origin, for use as genetic material fortransfer into rumen bacteria.
Other potential applications of these
W094/00578 ~139 ~9 PCT/AUg3/00307
23
cellulase cDNAs include transfer into some industrial
strains of microorganisms for more efficient conversion
of cheap plant material, even lignocellulosic wastes,
to commercially valuable products, such as ethanol,
butanol, acetic acid, citric acid and antibiotics. The
recombinant cellulases may also be used as a cellulase
source for industrial applications.
Industrial use of the recombinant cellulases celA and
cel D
Cellulase is one of the sixteen important
industrial enzymes. The current world market for these
enzymes is >750 million U.S. dollars with an annual
growth rate of 5-10% in volume. The potential use of
the recombinant celD enzyme is listed below:
1. To increase filtration rate of the beer in the
brewing industry. Cellulase is added to wort
to degrade ~-glucan which causes formation of
gels and hazes in beer and hence decreases
filtration rate of beer.
2. For waste water treatment in the pulp and
paper industry and starch industry. The
enzyme may be added to waste water to remove
cellulose residues in waste water recycling
processes. It may also be used to facilitate
drainage in paper making and the deinking of
newsprint.
3. For use in the dietary food, medicine and
cosmetic industries. Recent study has shown
that modified cellulose by partial enzymatic
depolymerisation was found to be a useful
product in these industries.
4. Other uses include clarification of fruit
juices, vegetable processing, bread making,
animal feed preparation and research purposes.
35 The use of celA and celD as qenetic material for
modification of some economicallY imPortant micro-
orqanisms for imProvement of cellulose utilisation
~13 q O 9 g I PCT/AU93/0030~
24
1. Modification of rumen bacteria for improvement
of plant fibre digestion by sheep and cattle.
2. Modification of silage inoculant bacteria
(lactic acid bacteria) to stimulate conversion
of cellulosic material to microbial protein
and increase nutritive value of silage as
animal feeds.
3. Modification of nitrogen-fixing microbes in
compost preparations or plant residues to
improve degradation of cellulosic material
which is used as energy to support the growth
of nitrogen-fixing bacteria.
4. Modification of ethanol-producing microbes
such as Saccharomyces cerevisiae or Zymomonas
mobilis for conversion of cellulosic material
such as agricultural wastes to ethanol for
industrial use.
The invention also includes within its scope
the following -
(i) DNA sequences derived from celA, celB, celC,
celD and celE cDNA clones;
(ii) DNA sequences derived therefrom (i) including
DNA sequences hybridisable therewith using a
standard hybridisation technique as described
in Sambrook et al. (1989);
(iii) celA, celB, celC, celD and celE enzymes having
the features described herein;
(iv) polypeptides having amino acid sequences
derived from celA, celB, celC, celD and celE
cDNA.
It will also be appreciated that the present
invention could also cover the following compositions:celA, celB, celC, celD or celE enzymes in
combination or mixtures of these enzymes as a pair,
triplet or as a mixture of four enzymes, and these
mixtures in combination with xylanase, eg. recombinant
xylanase derived from Neocallimastix patriciarum.
W094/00578 213 4~g I PCT/AU93/00307
Plasmid pCNP4.1 in E col i strain XL1-Blue has
been deposited at the International Depository
Australian Government Analytical Laboratories on June
~ 22, 1992 under accession number N92/27543.
Plasmid pCNP1 has been deposited at the
International Depository Australian Government
Analytical Laboratories on June 22, 1993 under
accession number N93/28000.
The term "essentially" as used herein refers
to 70-100% identity with the sequences shown in Fig. 13
and Fig. 15. The term "hybridise" as used herein
refers to a standard nucleic acid hybridisation
technique described by Sambrook et. al. (1989).
W094/00578 21~9 U9~ ~j PCT/AU93/00307
26
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CAVICCHIOLI, R. & WATSON, K. (1991). Molecular
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DEHORITY, B.A. (1991). Cellulose degradation in
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FLINT, H.J., McPHERSON, C.A. & BISSET, J. t1989).
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.
W094/00578 21 3 q ~ g ~ PcT/Aug3/on307
FLINT, H.J., McPHERSON, C.A. & MARTIN, J. (1991).
Expression of two xylanase genes from the
rumen cellulolytic bacterium Ruminococcus
~ flavefaciens 17 cloned in pUC13. Journal of
General Microbiology 137, 123-129.
FOONG, E., HAMAMOTO, T., SHOSEYOV, O. & DOI, R.H.
(1991). Nucleotide sequence and
characteristics of endoglucanase gene engB
from Clostridium cellulovorans. Journal of
General Microbiology 137, 1729-1736.
GILBERT, H.J., HAZLEWOOD, G.P., LAURIE, J.L., ORPIN,
C.G. & XUE, G.P. (1992). Xylanase A from the
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2072.
GILKES, N.R., HENRISSAT, B., KILBURN, D.G., MILLER,
R.C.JR. & WARREN, R.A.J. (1991). Domains in
microbial ~-1, 4-glycanases, sequence
conservation, function, and enzyme families,
Microbiological Review 55, 303-315.
GILKES, N.R., WARREN, R.A.J., MILLER, R.C.JR. &
KILBURN, D.G. (1988). Precise excision of
the cellulose binding domains from two
Cellulomonas fimicellulases by a homologous
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Journal of Biological Chemistry. 263, 10401-
10407.
GOMEZ DE SEGURA, B.G. & FEVRE, M. (1991). Cell wall
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GOYAL, A., GHOSH, B. & EVELEIGH, D. (1991).
Characteristics of fungal cellulases.
Bioresource Technology 36, 37-50.
W094/00578 ~1 3q 0 g ~ PCT/AU93/0030'
HAZLEWOOD, G.P., DAVIDSON, K., LAURIE, J.I., ROMANIEC,
M.P.M. & GILBERT, H.J. (1990). Cloning and
sequencing of the celA gene encoding
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strain A46. Journal of General Microbiology
136, 2089-2097.
HOWARD, G.T. & WHITE, B.A. (1988). Molecular cloning
and expression of cellulase genes from
Ruminococcus albus 8 in Escherichia coli
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LAKE, B.D. & GOODWIN, H.J. (1976). Lipids, In
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Edited by I. Smith and J.W.T. Seakins, Bath:
The Pitman Press.
LEVER, M. (1972). Anal. Biochem. 47, 273-279.
LI, X. & CLAZA, R.E. (1991). Fractionation
of cellulases from the ruminal fungus
Neocallimastix frontalis EB188. Applied and
Environmental Microbiology 57, 3331-3336.
MEINKE, A., GILKES, N.R., KILBURN, D.G., MILLER,
R.C.JR. & WARREN, R.A.J. (1991). Multiple
domains in endoglucanase B Journal of
Bacteriology 173, 7126-7135.
MESSNER, R. & KUBICEK, C.P. (1991). Carbon source
control of cellobiohydrolase I and II
formation by Applied and Environmental
Microbiology 57, 630-635.
ORPIN, C.G. & MUNN, E.A. (1986). Neocallimastix
patriciarum sp. nov., a new member of the
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ROBSON, L.M. & CHAMBLISS, G.H. (1989). Cellulases of
bacterial origin. Enzyme and Microbial
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~1~4099
W094/00578 ~ PCT/AU93/00307
-
29
ROMANTEC, M.P.M., DAVIDSON, K., WHITE, B.A., HAZLEWOOD,
G.P. (1989). Cloning of Ruminococcus albus
endo-~-1, 4-glucanase and xylanase genes.
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SAARILAHTI, H.T., HENRISSAT, B. & PALVA, E.T. (1990).
CelS: a novel endoglucanase identified from
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SAUL, D.J., WILLIAMS, L.C., GRAYLING, R.A., CHAMLEY,
L.W., LOVE, D.R. & BERGQUIST, P.L. (1990).
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TAYLOR, K.A., CROSBY, B., McGAVIN, M., FORSBERG, C.W. &
THOMAS, D.Y. (1987). Characteristics of the
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Bacteroides succinogenes expressed in
Escherichia coli. Applied and Environmental
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W094/00578 ~ ~ 3 ~ o g 9 PCT/AUg3/0030-
TEATHER, R.M. & WOOD, P.J. (1982). Use of Congo Red-
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TOMME, P., VAN TILBEURGH, H., PETTERSSON, G., VAN
DAMME, J., VANDEKERCKHOVE, J., KNOWLES, J.,
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BHIKHABHAI, R. & PETTERSSON, G. (1986).
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XUE, G.P. & MORRIS, R. (1992). Expression of the
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XUE, G.P., ORPIN, C.G., GOBIUS, K . S ., AYLWARD, J.H. &
SIMPSON, G.D. (1992). Cloning and expression
of multiple cellulase cDNAs from the anaerobic
rumen fungus Neocallimastix patriciarum in
Escherichia coli. Journal of General
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W094/00578 ~134 9 9 1"~ PCT/AU93/00307
31
YAGUE, E., BÉGUIN, P. & AUBERT, J.-P. (1990).
Nucleotide sequence and deletion analysis of
the cellulase-encoding gene celH of
Clostridium thermocellum. Gene 89, 61-67.
W0 94/00578 Q ~ 3a~ 0 ~ 9 PCI /AU93/0030~
32
Tab1e1 Activity of the cioned cellulases on various substrates.
Specific activity
Slubstrate (nmol product min ' (mg protein)~' )
celA celB celC ceID ce~E
CM-cellulose 466 54 21 4929 1256
Avicel 196 1.4 0.9 179 50
Amorphous 1874 6.2 20 812 ND
cellulose
Xylan 0 0 0 466 124
Lichenan 13600 ND ND 14312 ND
pNPG 0
pNPC 0 1.3 0 169 80
MUG ND- ND ND 0 ND
MUC ND ND ND 944 150
ND- Not Determined
Crude cell Iysate preparations were used for the measurement of
enzyme activities as described in Methods. The values given are
csentative of at least three assays and are e..p,cised as nmol
product (reducing sugar or ~nitrophenol released) min~' (mg
protein)~ ' .
WO 94/00578 . PCI`/AU93/00307
- 21~9099
Table 2
Effect of celA and celD enzymes and pNX-Tac xylanase in combination onhydrolysis rate of natural plant material
Relative rate of hydrolysis
control 0.056
pNX-tac Xylanase 1.07
CelA and CelD enzymes
plus pNX-Tac xylanase 2.31
The rate of plant fibre hydrolysis was measured by assaying reducing sugar
production
by the method of Lever (1972).
The hydrolysis reaction was performed at 40 C for 2 days using Setaria stem within vivo digestibility of 64.7%.
W094/00578 2 1~ ~ 9 9 PCT/AU93/0030'
34
LEGENDS
Figure 1 - Restriction maps of CelD and CelE.
Abbreviations for restriction enzymes:
E,EcoRI; B,BglII; K,KpnI; P,PvuII, X,XhoI; D,DraI.
Figure 2 - Restriction maps of celA, celB and celC.
Figure 3 - Cross-hybridization of three cellulase cDNA
inserts by Southern blot analysis. Plasmids containing
celA (A), celB (B) and cel C ( C ) were cut with EcoRI and
XhoI ( the cDNA cloning sites) and fractionated on 1~
(w/v) agarose gel. Digoxigenin-labelled RNA probes
generated from 3'-region-deleted cDNA clones were used
for hybridization:celA' probe, left blot; celC' probe,
right blot. Large arrows indicate the cDNA inserts
being hybridized. The bands indicated by small arrows
are the cloning vector being hybridized, as the RNA
probes contain part of the sequence from the vector.
Numbers on the margins indicate the sizes, in kb, of
molecular markers (BstEII fragments of ~DNA).
Figure 4 - Congo-red staining assay of the celD enzyme
activity on lichenan and laminarin. Two microlitres of
crude enzyme extract were placed onto wells cut in the
agarose plates containing lichenan or laminarin as
described in Methods. After incubation at 39C for 2
hr and staining with Congo red, hydrolysis of
substrates is indicated by presence of a yellow halo in
the red background around the well.
Figure 5 - Analysis of products of cellulosic compounds
hydrolysed by the celD enzyme. (a) Crude cell lysate
was prepared from E. coli harbouring plasmid pCNP4.1
and low-molecular-mass compounds were removed by spin-
dialysis using a Centricon-10 tube (Amicon). The
enzyme preparation was incubated with cellodextrins:
[cellotriose (G3), cellotetraose (G4) and cellopentaose
SUBST~UTE SHEET ¦
W094/00578 213 q O 9 9 i PCT/AU93/00307
-
(Gs)~ each 2 mg ml~'] or with 1% (w/v) Avicel (C) as
described in Methods. Partial hydrolysis of
cellodextrins is shown to illustrate the intermediate
- products. Hydrolysis products were identified by their
~f values on a TLC plate. Authentic cellodextrins (S)
- are shown in the rightmost lane and the positions of
glucose (Gl), cellobiose (G2), G3 and G4 are indicated.
(b) Illustration of the catalytic mode of the celD
enzyme on cellotetraose.
Figure 6 - Analysis of hydrolysis products of the celA,
celB and CelC enzymes on cellulosic compounds. (a)
Crude cell lysates were prepared from E. col i
harbouring recombinant plasmids ( celA, celB and celC)
and the small molecules were removed by spin-dialysis
using Centricon-10 tubes (Amicon). The enzyme
preparations were incubated with cellodextrins (2 mg
ml~'); cellotriose (G3), cellotetraose (G4) and
cellopentaose (G5) or with 1% (w/v) Avicel (C) as
described in Methods. Products were identified by TLC.
Complete hydrolysis of cellodextrins by the celA enzyme
and partial hydrolysis by the celB and celC enzymes are
shown. Authentic cellodextrins (S) are shown in the
rightmost lane. (b) Illustration of the catalytic
mode of the three cloned enzymes on cellotetraose.
Figure 7 - Effect of pH on the activity of the
recombinant cellulases. Cellulase assays were
performed at 40C in 50 mM Na-citrate (pH4-7) or 25mM
Tris-C1/50mM NaCl (pH7.5-9.5) containing 1% Avicel for
2 2 hours.
Figure 8 - Effect of incubation temperature on the
activity of the recombinant cellulases. Cellulase
assays were performed in 50 mM Na-citrate (pH 5 or 6)
containing 1% Avicel for 2? hours.
pH5.-*-.;pH6 -.-
¦ SU~5S~ JTE S5~EET
W094/00578 ~ 3 9 ~ 9 9 PCT/AU93/00307
36
Figure 9 - Time course of cellulase hydrolysls by the
cloned celA enzyme. Cellulase hydrolysis was performed
at 39C in 50mM Na-citrate (pH6.0) containing 1%
Avicel. The celA enzyme (1-12~L) was added to the
reaction of a final volume of 500~1.
Figure 10 - Time course of cellulose hydrolysis by the
cloned celD enzyme. Cellulose hydrolysis was performed
at 39C in 50mM Na-citrate (pH6.0) containing 1%
Avicel. The celD enzyme (1-12~L) was added to the
reaction of a final volume of 500~L.
Figure 11 - Effect of celA and celD enzymes in
combination on the rate of crystalline cellulose
hydrolysis. Cellulose hydrolysis was performed at 39C
in 50mM Na-citrate (pH6) containing 1% Avicel for 4 hr.
Figure 12 - Restriction map of celD cDNA and its
deletion mutants. The positions of the cleavage sites
of EcoRI(E), Bg/II(B), KpnI(K), ~auII(P) and XhoI(X)
are shown. The positions of deletion mutants of celD
are indicated by solid bars and numbers in kbp
corresponding to the positions in pCNP4.1. The enzyme
activity of the clones was determined on substrate-
containing agarose gel plates and cellulose-binding
capacity was determined with Avicel: +, active, -,
inactive; ND, not determined, CMC, CM-cellulose; Xyn,
xylan; Av, Avicel: CB, cellulose-binding.
¦ SU~5TiTUT~ S!5~T ¦
W094/00578 ~1 3 q ~ 9 9 ~ ~ , i PCT/AU93/00307
37
Figure 13 - Characterisation of Neocallimastix
patriciarum celD nucleotide sequence.
"A" - Single base induces frame shift and TAA stop
codon
"B" - Apparent original TAG stop codon.
~ N-terminal of ~-galactosidase ~-
359 amino acid catalytic cellulase
domain.
Catalytic domains present in
triplicate.
~ Predicted amino acid identity of each
catalytic domain is >95~.
Catalytic domains show most identity
with sub-family A4 cellulases (A4
comprises only endoglucanases from
anaerobic bacteria, including
Butyrivibrio, Prevotella and
Ruminococcus).
Serine-, threonine- and proline-rich
~ linker sequence separating each of the
catalytic domains.
Cysteine-rich repeats of unknown
function. Repeats show limited
~ identity with analogous carboxy-
terminal domains encoded by
- Neocallimastix patriciarum xynA cDNA.
AT-rich 3' untranslated sequence and
polyA transcription terminator.
Figure 14 - pNX-Tac construct
~ SU~STI, UT~ ET
W094/00578 21 3q 0 ~ 9 PCT/AU93/0030
38
Reproduced hereinbelow are sequence listings wherein -
SEQ ID NO:1 refers to nucleotide sequence ofNeocallimastix patriciarum celD cDNA. The sequence
underlined is derived from pBluescript SK-vector and
the EcoR1 adaptor used for cDNA cloning.
SEQ ID NO:2 refers to translated sequence of domains I
and II of Neocallimastix patriciarum celD cDNA.
Translated polypeptide includes the N-terminus of the
~-galactosidase ~-peptide (derived from nucleotides
1-111) and amino acids derived from the 5~
oligonucleotide linker (nucleotides 112-124) used in
cDNA library construction.
SEQ ID NO:3 refers to translated sequence of domain III
of Neocallimastix patriciarum celD cDNA.
SEQ ID NO:4 refers to the sequence of the modified
xylanase cDNA in pNX-Tac.
¦ SUBSTITUTE SI;EE
.213q~gg . ,
W O 94/00578
~I~ PC~r/A U93/00307
Ce1D
1 .~TCACCATGA TTACGCCAAG CTCGAAATTA ACCCTCACTA AAGGGAACAA AAGCTGGAGC
61 ~C~CCGCGG ~G~ ~GC TCTAGAACTA GTGGATCCCC CG~OCl~CAG GAATTCGGCA
121 C_ CTCCAA TCCGTGATAT TTCATCCAAA GAA~TAATTA AAGAAATGAA l11CGC11~G
181 AATTTAGGTA ATACTITAGA TOCTCAATGT ATTGAATACT TAAATTATGA TAAGGATCAA
241 A.-~ -~G AAACTTGCTG GGGTAATCCA A`AGACTACTG AAGATATGTT CAAGGTTTTA
301 ATGGATAACC AATTTAATGT ~1~CCG1ATT CCAACTAC~T ~-OG-~A C1 .C~-1~AA
361 GCTCCAGATT ACAAGATTAA TGAAAAATGC TTAAACAGAG IICATCAAAT TGTTGATTAT
421 CCATAC~AAGA ATCGAGC m C~ALC~A AATCTTCACC ATGAAACTTG GAACCATCCC
481 1 ~ AAA CICTTGAC~C TY CCAAGGAA ATCTTAGAAA A~A1~GG~C TCAAATTGCT
541 AAAGAATTTA AG~A--A.GA TGAACACTTA ATTTTTGAAG GATTAAACGA ACCAACAAAG
601 AATGATACTC CAC~TGAATG GAC-CC-~- GATCAACAAG CATGGGATGC TGTTAATGCT
661 ATGAATGCCG ~ AAA GAC~A~CS- AG11~1GG~ GTAATAATCC AAAGCC~AT
721 C~TATGATCC C~C~A~AIaC ~ ~l AATGAAAATT CATTCAACAA CTTTATTTTC
781 CCAGAAGATG AToAC;AGCT A-1OC11~- GTTCATGCIT A-G~-CCATA CAA~1--GCC
8q1 TTAAATAATG G~AAGGAGC TGTTGATAAG 1--GA~GC~G C~GGTAAGAA AGATCTTGAA
901 TGGAACATTA A~ 1AATGAA GAAGAGA m Gl1GA~AAG C~A1~ AT GA1~11GC1
961 GAA~h~G-~ CCATGAATCG TGATAATGAA GAAGATCGTG C~AGC11G~GC TGAATTCTAC
1021 ATGCAAAhGG TCAL1~C1AT GGGAGTTCCA CAAGrCTGGT GGGATA~TGG TATCTTTG~AA
1081 GGTACCGGTG AAC~l-ll~ AT CGTAAAAACT TAAACATTGT TTATCCAACT
1141 A-C~11~1~ CTTTACAAAA GGCAAGAGGT TTAGAAGTCA AlGl-lCllCA TGCTATTGAA
1201 Aa~AAA~cAG AAGaACCAAC TAAAACTACT GAACCAGTTG AACCAACTGA AACTACTAGT
1261 ccAoaAoAAC CAGCIGAAAC TACTAATCCA GAAGAACCAA C~GGTAATAT TCGTGATATT
1321 1~-~AGG AATTAATTAA AGAAATGAAT 11C~C11~A ATITAGGTAA TAC~TAGAT
1381 GC~CAATGTA TTGAATACTT AAA11A1GAI AAGGATCAAA C1GC11~1GA AA 11GC-GG
1441 GGTAATCCAA ACACTACTGA AGA-h1C11C AAGGTTTTAA TGGATAACCA ATTTAATGTT
1501 11~C~1A11~ CAACTACTTG ~1C1G~AC llC~GlGAAG CTCCACATTA CAAGATThAT
1561 GA`AAAATG'GT TAAAGAGACT TCATGAAATT ~11~A11ATC CATACAACAA TGGAGCTITC
1621 ~11AIC11AA ATCrTCACCA TGAAAC`TTGG AAC~Al~11 TCrCrGaAAC TCTTGACACT
1681 GCCAAGGAAA TTITAGAAAA GA~lG~l~l CM ATTGCTG AAGAATTTAA GGATTATGAT
1741 GAACACTTAA TlSTTGaAGG ATTAAAQGAA CCAAGAAAGA ATGATAC~CC AGTTGAATCC
1801 AL1~1~G1G ATCAAGAAGG A1G~GA~GC1 GTTAATGCTA TGAATGC~GT TTTCTTAAAG
1861 AC1~11~1A ~11~1G~1G~ TAATAATCCA AAG~ A1C 11Al~AnCCC 1~_ArA1~L1
1921 GC~G~ll~lA ATGAAAATTC ATTCAAGAAC lllAllll~C CAGAAGATGA TGACAAGGTT
1981 A11~C11~1G 11~A1G~11A GC-CCA1..C A~L-11G~C- TAAATAATGG TGAAGS;OCT
2041 GTTGATAAGT 11~A1G W ~` TGGTAAGAAT GACCTTGAAT GGAATATTAA CTThATGAAG
2101 AAGAGATTTG TTGATCAAGG TATTCCAATG All~l1G~G AATATGGTGC CATG MTCGT
2161 GATAAT~AAG AACA1C~1GC AGC11G~GC1 GAATTCTACA CGAUAAGGT CA~1GC1AIG
2221 GG`AGTTCCAC AAG1~C-G GGATAATGGT ATCTTTGAAG GTAC~ rGA AC~ Gl
2281 ~11~11~Al~ GTAGAAACTT AAA~A11~11 TATCCAACTA Y~11GClGC TTTACAAAAG
2341 GGAAGAGGTT TAGAAGTCAA 1~11~11~AT G-1~11~AAA AAAAAACCAG AAGAACCAAC
2401 TAAGACTACT GAACCAGTTG AACC;~C1GA AACTACTAGT CCAGAAGAAC C`AACTGAAAC
2461 TACTAATC~A GAAGAACCAA CCGGTAATAT TCGrGATATT TCATCTAAGG AATTAATTAA
2521 AGAAATGAAT 11C~11GGA ATTTAGGT M TACTTTAGAT GCTC M TGTA TTGAATACTT
2581 AAA11A1Ghl AAGGATCAAA C1~11~1GA hhL-11V~1~G GGTAATCCAA AGACTACTGA
2641 AGA1A1~11C M GGTTTTAA TGGATAACCA ATITAATC~T 11~1A11~ CAACTAC~TG
2701 C1~ ~AC -C~C1~AG CTCCAGATTA CAAGATTAAT GAAA M TGGT TAAAGAGACT
2761 TCATGA M TT ~11GA~1~ CATACAAG M TGGAGCTTTC C11A~11AA ATCTqCACCA
2821 TGAAAC$TGG AAC~A~G~11 lC~C~C~AC TCrTCDCACT GCCAAGGAAA ITTTAGAAAA
2881 GA1--~1C1 CAAA`1~ ~ AAGAATTTAA ~GA11AIGAT GAACAC~T M TTTTTGAAGG
2941 ATTAAACGAA CCMGA~AAGA ATGATACTCC AGTTGMTGG AC1~1G~1C ATC MGAAGG
3001 A1G~3~A-1GC1 GTTAATGCTA TGAATGCCGT TrTCITAAAG A~A1~CG1A ~11~1G~1GG
3061 TAATAATCCA AAG~G1~Ar~ TTATGATCCC ~AlA1~1 GL1~C11~1A ATGAAAATTC
3121 ATT~AAGAAC 11~A1111CG CACAAGATGA TGACAAGGTT AllGL1l~lG l~A~GC~lA
3181 1~L1C~ATAC AAC111GL~81 TAAATAATGG TGCAGGAGCT GTTGAT MGT ~1CAr~CGG
3241 TGCTAACA M CATCTTGAAT GGAACATTAA CTTAATGMG AAGAGATTTG TTGATCAAGG
3301 lAl 1CC~ATG Al 1~11~G1G AATATGGTGC CATGAACC~GT CATAAT~AAG AAGAACGTGC
3361 TACATGGGCT GAATTCTACA TæAAAAGGT C~ACTGCTATG GCAGTTCCAC AAG1~1~GLG
3421 C~ATAATGGT ~1~111~AAG GTACCGGIGA AL~1111~1 ~11~11 ~ATC G~AAAAAC~T
3481 AAAGATTGTT TATC~ACTA 1~11~1GC TTTACAA MG GGMGAGGTT TACAAGITAA
3541 G~11C11CAT G~AAATGAAG AACAAACAGA AGAATGTTGG TCTGAA MGT ATGGTTATGA
3601 Al~-ll~ll~l C~TAACAATA CTAAGGT~GT AGTCAGTGAT GAAAGTGG M A11~GGG1~1
3661 TG~AAATGGT AA11G~1~1G Cl~11~11~A ATACACTGAA AAA~11G~1 CACTTCCATT
- 3721 TGGATACCCA '1~11C1 AC ATTGTAAGGC TCTTACTAAG 6ATGAAAATG GTAAATGCGG
3781 AGAAGTAAAT GGTGAATGGT ~l~lAl~Cl TG~TGATAAA TGITAGATTA TAAAATAAAA
3841 ATAAATAGAT ll~l~AlGA AAATTATTAA TGAATAATAA ATAAAATAGA AAATTTTATA
3901 TAAACATATT TCTAATA~AA 6ATCTAATTA TGTATTTTTT 6-1~11ATT CITTCAAATA
- 3961 AAAAAAGTAA GÆAAAGAAAA TATATAAAAT AAAAAAAAAA AATAAATAAA TAAATATTTT
4021 AATTATTTTT TTTTTA(`.TAA AAAAAA~AA TTTAATTAAA ATATAATTAA AA('TAAAAAA
SEQ ID NO:1 `~~-
SUB~i T ~ JTE SffEET ~
WO 94/00578 ~ - PCI/AU93/00307
213qO99
40 .
celD
4081 AAAAA~ AACI~G
SEQ ID N0: 1
¦ SlU~ JTE SHEET ~
WO 94/00578 21 3 q ~ ~ ~ PCI`/AU93/00307
` F ~
celD doml/2 Tran~lated sequence
S~-~n~e Range: 1 to 2400
10 20 30 40 50
ATG ACC ATG ATT ACG C'CA AGC TCG AAA TTA ACC CTC ACT AAA GGG AAC AAA AGC
TAC TGG TAC TAA TGC GGT TCG AGC TIT AAT TGG GAG TGA TTT CCC TTG TIT TCG
M T M I T P S S K L T L T K G N K S>
a_a a_a_a T~NCr~TIC~ OF CELD DOMl/2 a a a a a_~
100
.
TGG AGC TCC ACC GCG GTG GCG GCC GCT CTA GAA CTA GTG GAT CCC CCG GGC TGC
ACC TCG AGG TGG CGC CAC CGC CGG CGA GAT CIT GAT CAC CTA GGG GGC CCG ACG
W S S T A V A A A L E L V D P P G C>
_a_a_a_a a _TRANSLATIaN OF CELD D0Ml/2 a a_a a a >
110 120 130 140 150 160
~ ~ ~ .
AGG AAT TCG GCA C'GA GCT CCA ATC CCT GAT ATT TCA TCC AAA GAA TTA ATT AAA
TCC TTA AGC C'GT GCT CGA GGT TAG GCA CTA TAA AGT AGG TIT CTT AAT TAA TTT
R N S A R A P I R D I S S K E L I R>
a_a_a_a~TEtANSL~TI~ OF CELD DOt~lt2 a_a a a_a_>
170 180 190 200 210
GAA ATG AAT TTC GGl IGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA
CTT TAC TTA AAG C'CA ACC TTA AAT CCA TTA TGA AAT CTA CGA GIT ACA TAA C'TT
E M N F G W N L G N T L D A Q C I E~
a a_a a_a _TRANS ~TICN OF CELD D0Ml/2_a a a~_a_a_>
220 230 240 250 260 270
TAC TTA AAT TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT C'CA
ATG AAT TTA ATA CTA TTC CTA GIT TGA CGA AGA CTT TGA ACG ACC C'CA TTA
Y L N Y D R D Q T A S E T C W G N P>
a a a a a _TRANSLATICN OF CELD D0Ml/2 a a_a a a_>
280 290 300 310 320
AAG ACT ACT GAA GAT ATG TTC AAG GIT TTA ATG GAT AAC CAA TTT AAT GIT TTC
TTC TGA TGA CTT CTA TAC AAG TTC CAA AAT TAC CTA TTG GIT AAA TTA CAA AAG
K T T E D M F K V L M D N Q F N V F>
_a a a_a a _TRANSI~ATICN OF CELD D0Ml/2 a_a a a a >
330 340 350 360 370
--
CGT ATT CCA ACT ACT TGG TCT GGT CAC TTC GGT GAA GCT CCA GAT TAC AAG ATT
GCA TAA GGT TGA TGA ACC AGA CCA GTG AAG CCA Cl~ CGA GGT CTA ATG TTC TAA
R I P T T W S G H F G E A P D Y K I>
a a_a_a~rR~NCr~TIat7 OF OE LD D0t51/2 a a a a_a_>
380 390 400 410 420 430
~
AAT GAA AAA TGG TTA AAG AGA GIT CAT GAA ATT GTT GAT TAT CCA TAC AAG AAT
TTA CTT TTT ACC AAT TTC TCT CAA GTA CIT TAA CAA CTA ATA GGT ATG TTC TTA
N E K W L K R V H E I V D Y P Y K .N>
_a_a_a a a _TR~NCr~TION OF CELD D0tSl/2_a a a_a_a_>
440 450 460 470 480
~ . ~ ~ .
GGA GCT TTC GIT ATC TTA AAT CTT CAC CAT GAA ACT TGG AAC CAT GCC TTC TCT
CCT CGA AAG CAA TAG AAT TTA GAA GTG GTA C,TT TGA ACC TTG GTA CGG AAG AGA
G A F V I L N L H H E T W N H A F S>
a_a_a a a_TRANSLPTION OF CELD D0Ml/2_a_a_a_a a_>
490 soo 510 520 530 S40
SEQ ID NO:~
~ SUe~g~ JTE SHEET ¦
WO 94/00578 2 1 3 q ~ 9 9 Pc~r/Aug3/oo307
ceLD doml/2 Translate~ Sequence 4 2
GAA ACT CTT GAC ACT GCC AAG GAA ATC TTA GAA AAG ATT TGG TCT CAA ATT GCT
CTT TGA GAA CTG TGA CGG TTC CTT TAG AAT CTT TTC TAA ACC AGA GTT TAA CGA
E T L D T A K E I L E K I W S Q I A,
_ a _ a _ a _ a _ a _TRANSLATION OF CELD DOMlt2 a a a _ a a
550 560 570 580 590
AAA GAA TTT AAG GAT TAT GAT GAA CAC TTA ATT TTT GAA GGA TTA AAC GAA CCA
TTT CTT AAA TTC CTA ATA CTA CTT GTG AAT TAA AAA CTT CCT AAT TTG CTT GGT
K E F R D Y D E H L I F E G L N E P~
_a a _ a _ a a _ TRANSLATION OF CELD DOMl/2 a a a a a _
600 610 620 630 6g0
AGA AAG AAT GAT AiCT CCA GTT GAA TGG ACT GGT GGT GAT CAA GAA GGA TGG GAT
TCT TTC TTA CTA TGA GGT C M CTT ACC TGA CCA CCA CTA GTT CTT CCT ACC CTA
R K N D T P V E W T G G D Q E G W D>
a _ a_a_ a a_ TRANSLATION OF CELD D0Mlt2 aaaa a _
650 660 670 680 690 700
~ . . ~ . ~
GCT GTT M T GC,T ATG AAT GCC GTT TTC TTA AAG ACT ATT CIGT AGT TCT GGT G
C~GA CAA TTA CGA TAC TTA CGG CAA M G M T TrC T~A TAA GCA TCA AGA CCA CCA
A V N A MN A V F L K T I R S S G G>
_ a a a _ a a _TRANSLATION OF CELD DOMl/2 a a a a a _ >
710 720, 730 740 750
~ ~ . ~ .
AAT AAT CCA AAG CGT CAT CTT ATG ATC CCT CCA TAT GCT GCT GCT TGT AAT GAA
TTA TTA GGT TTC GCA GTA GAA TAC TAG GGA GGT ATA CGA CGA CGA ACA TTA CTT
N N P K R H L M I P P y A A A C N E~
a. a a a a_ TRANSLATION OF CELD D0ffl/2 aa_ a _ a _a_>
760 770 780 790 800 810
~ ~ .
AAT TCA TIC AAG AAC TTTATTTTC CCA GAA GAT GAT GAC A~G GTT ATT GCT TCT
TTA AGT AAG TIC TrG AAA T M AAG GGT CTT CTA CTA CTG TIC C M T M CGA AGA
- N S F K N F I F P E D D D K V I A S>
a a_ a _ a a_TRANSLATION OF CELD DOMl/2 a a aa a _ >
820 830 840 850 860
~ ~ ~
GTT C'AT GCT TAT GCT CC~ TAC AAC TTT GCC TTA M T M T GGT GAA ~ GCT GTT
GAA GTA CGA ATA CGA GGT ATG TTG AM CGG M T TTA TTA CCA CTT CCT CGA CAA
V H A Y A P Y . N F A L N N G E G A V~
a a _ a a a_ TRANS~ATION OF CELD DOMl/2 a a _ a a a _
870 880 890 900 910
GAT AAG TrT GAT GCT GCT GGT AAG M A GAT CTT GAA TCG AAC ATT M C TTA ATG
CTA TTC AAA CTA CGA CGA CCA TTC TTT CTA GAA CTT ACC TTG $AA TTG AAT TAC
D X F D A A G K K D L - E W N I N L M~
a a a _ a a_ TRANSLATION OF ~~Frn D0Ml/2 a a a a a _
920 930 940 950 960 970
AAG AAG AGA TTT GTT GAT CAA GGT ATT CcA ATG ATT cTT GGT G~A T~T GGT GCC
TTC TTCTCT AAA CAA CTA GTT C'CA TAA GGT TAC TAA GAA CCA crT ATA CCA CGG
K R R F V D Q G I P M I L G E Y G A>
_ a _ a_aa_ TRANSLATION OF ~rn D0Ml/2 _ aa_ a _a a _
980 990 1000 1010 1020
ATG AAT CG$ GAT AAT GAA GAA GAT CGT GCA GCT TGG GCT GAA TTC TAC ATG GAA
TAC 'TTA GCA CTA TTA CTTCTT CTA GCA CGT CGA ACC CGA CTT AAG ATG TAC CTT
M N R D N E E D R A A W A E F Y M E>
aaa_aa_ TRANSLATION OF CELD D0M1/2 a a _ aa_ a _
SEQ ID NO:2
~ SUB8TITUTE 8HEET ~
WO 94/00578 ~, ~ . PCI`/AU93/00307
-
celD doml/2 Tr;~ncl ~Itffl Se~auence 4 3
1030 10gO 1050 1060 1070 1080
tr ~ ~
AAG GTC ACT GCT ATG GGA GIT CCA CAA GTC TGG TGG G~T AAT GGT ATC TTT GAA
TTC CAG TGA CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA cTr
K V T A M G V P Q V W W D N G I F E>
a_a_a a_a TRANSLATICN OF CELD DOM1/2 _a a a a~a_>
1090 ~00 1110 1120 1130
~ . ^ . ~
GGT ACC G5T GAA CGT TTT GGT CIT CTT GAT CGT AAA AAC TTA AAG ATT GTT TAT
CCA TGG CCA CTT GCA AAA CCA GAA GAA CTA GCA TTT TTG AAT .1~ TAA CAA ATA
G T G E R F G L L D R K N L K I V Y>
a_a_a a_ a_ TR ~ LATI~ OF OELD D0t~11/2 a a a a_a_>
1140 llS0 . 1160 1170 1180
.. .. . .
CCA ACT ATC GTT GCT GCT TTA CAA AAG GGA AGA G~T TTA GAA GTC AAT GTT GTT
GST TGA TAG CAA CGA CGA AAT GTT TTC CCT TCT CCA AAT C~- CAG TTA CAA CAA
P T I V A A L Q K G R G L E V N V V>
a_a_a_a_~ TRANSLATI~ OF OELD D0MV2-a a a a_~ >
1190 1200 1210 1220 1230 1240
.. .
CAT GCT ATT GAA AAA AAA CCA GAA GAA CCA ACT AAA ACT ACT GAA CCA GTT GAA
GTA CGA~rAA CTT TTT TTT .T CTT CTT GGT TGA TTT TGA TGA CTT GGT CAA CTT
H A I E K K P E E P T ~ T T E P V E>
a a_a a_aT~tA~TION OF CELD D0M1/2_a a a_a_a_~
1250 1260 1270- 1280 I290
~ .
CCA ACT~GAA ACT ACT AGT CCA GAA GAA CCA GCT GAA ACT ACT AAT CCA GAA GAA
GGT TGA CTT TGA TGA TCA GCT CTT CIT oeT CGA CTT TGA TGA TTA GGT CTT CTT
P T E T T S P E E P A E T T N P E E>
a a a a_a~TRANSl~TIC~ OF OELD DOM1/2 a a a a a_>
1300 1310 1320 1330 1340 1350
CCA A GGT AAT ATT CGT GAT ATT TCA TCT AAG GAA TTA A~T AAA GAA ATG AAT
GGT TGG CCA TTA TAA GCA CTA TAA AGT AGA l-C CTT AAT TAA TTT CTT TAC TTA
P T G N I R D I S S ~ E L I K E M N>
a_a_a_a_a ~`'~TI~LD ~1/2 a_a a a_a_>
1360 1370 1380 1390 1400
TTC GGT TGG AAT TTA GGT AAT ACT TTA GAT GCT CAA TGT ATT GAA TAC TTA AAT
AAG CCA ACC TTA AAT CCA TrA TGA AAT CT~ CGA G~T ACA TAA CTT ATG AAT TTA
F G W N L G N T L D A Q C I E Y L N>
a a_a a_a n2uLn~TIcN OF CELD D4M1/2 a a a_a_a_>
1410 1420 1430 1440 14S0
~ ~ ~
TAT GAT AAG GAT CAA ACT GCT TCT GAA ACT TGC TGG GGT AAT CCA AAG ACT ACT
ATA CTA TTC CTA GTT ~GA CGA AGA CTT TGA ACG ACC CCA TTA GGT ITC TGA TGA
Y D K D Q T A S E T C W G N P K T T>
a ~ ~ ~_~.T~TI~OFOE~/2 a_~ a_>
1460 lq70 1480 1490 1500 1510
GAA GAT ATG TTC AAG GTT TTA ATG GAT AAC CAA T~T AAT GTT TTC CGT ATT CCA
CTT CTA TAC AAG TTC CAA AAT TAC CTA TTG GTT AAA TTA CAA AAG GCA TAA GGT
E D M F X V L M D N Q F N V F R I P>
a a ~ a_a_ TR~TIC~N OF CELD DOtS1/2_ a a_a_a_a_>
1520 1530 lSg0 1550. lS60
~
. ACT ACT TGG TCT GGT CAC TrC GGT GAA GCT CCA GAT TAC AAG ATT AAT GAA AAA
SEQ ID NO:2
i ;3UBSTITUTE 8HEET ~
W O 94/00578 - PC~r/A U93/0030-
213~09g `
: ` 44
celD doml/2 TranslaLed Sequence
TGA TGA ACC AGA CCA GTG AAG CCA CTT CGA GGT CTA ATG 1~ TAA TTA crr TTT
T T W S G H F G E A p D Y K I N E K>
a a a a_a_TRA~SLATION OF CELD DOM1/2_a_a a a a_~
1570 lS80 1590 1600 1610 1620
~ , . . . .
TGG TTA AAG AGA GTT CAT GAA ATT GTT GAT TAT CCA TAC AAG AAT GGA GCT TTC
ACC AAT TTC TCT CAA GTA CTT TAA CAA CTA ATA GGT ATG TTC TTA CCT C(~A MG
W L K R V H E I V D y p y K N G A F>
a_a a aa_ TRANSLATION OF CELD D0Ml/2 a a a_aa>
1630 1640 1650 1660 1670
~ ~ ~
GTT ATC TTA AAT CTT CAC CAT GAA ACT TGG AAC CAT GCT TTC TCT GAA ACT CTT
CAA TAG AAT TTA GAA GTG GTA CTT TGA ACC TTG GTA CGA AAG AGA CTT TGA GAA
.VIL N L H H E T W N H A F S E T L>
_a_aa_a_a _TRANSLATION OF~ErD DOMl/2 _ aa_aaa>
1680 1690 1700 1710 1720
~ ~ . ^ .
GAC ACT GCC AAG GAA ATT TTA GM AAG ATT ~;G TCT CAA ATT GCT GAA GM TTT
CTG TGA CGG TTC CTT TAA MT CTT TTC TM ACC AGA GTT TAA CGA CTT CTT AAA
D T A X E I L E K I W S Q I A E E F>
aaaaa _TRANSLATION OF CELD DOM1~2 a_aaaa_>
1730 1740 17S0 1760 1770 1780
~ ^ --
AAG GAT TAT GAT GAA CAC ITA ATT TTT GM GGA TTA AAC GAA CCA AGA AAG AAT
TTC CTA ATA CTA CTT GTG AAT TAA MA CTT CCT AAT TTG CTT GGT TCT TTC TTA
R D Y D E H L I F E G L N E P R R N>
aaaa_a_ TRANSLATION OF CELD DOMl/2 aaaaa_>
1790 1800 1810 1820 1830
GAT ACT CCA GTT GAA TCC ACT GGT GGT GAT CM GAA GGA TGG GAT GCT GTT AAT
CTA TGA GGT CAA CTT AGG TGA CCA CCA CTA GTT CTT CCT ACC CTA CGA CM TTA
DTPVEST G G D Q E G W D A V N>
aaaaa _TRANSLATION OF CELD D0Ml/2 aaaaa>
18q0 1850 1860 1870 1880 1890
GCT ATG AAT GCC GTT TTC TTA AAG ACT ATT CGT AGT TCT GGT GGT AAT MT CCA
CGA TAC TTA ~ CM AAG AAT TTC TGA TAA GCA TCA AGA CCA CCA ~TA TTA GGT
A M N A V F L K T I R S S G G N N P~
a_a_aaa_ TRANSLATION OF CELD D0Ml/2. a aa_a_a>
1900 1910 1920 1930 1940
~ ~ .
AAG CGT CAT CTT ATG ATC CCT CCA TAT GCT GCT GCT TGT AAT GAA AAT TCA TTC
TTC GCA GTA GM TAC TAG GGA GGT ATA CGA CGA CGA ACA TTA CTT TTA AGT MG
K R H L M I P P Y A A A C N E N S F>
aaa_aa_ TRANSLATION OF CELD D0Ml/2 a_ aaaa>
1950 1960 1970 1980 1990
~ ~ --
AAG AAC rrr ATI' ~C CCA GAA GAT GAT G~C AAG Grr ATI GCT TCT GTT CAT GCT
TTC ~ AAA TM MG GGT CTT CTA CTA CTG TTC CAA TAA CGA AGA CAA GTA CGA
K N F I F P E D D D K V I A S V H A>
aaa a a _TRANSLATION OF CELD DOMlt2 _ a_ a_a_a a_,
20002010 2020 2030 2040 2050
~~ ~ --
TAT GCT CCA TAC MC TIT GCC TTA MT AAT GGT GAA GGA GCT GTT GAT AAG TTT
ATA CGA GGT ATG Tl~, MM C~;G AAT TTA TTA CCA CTT CCT CGA CAA CTA TTC A~A
Y . A P Y N F A L N N G E G A V D ~C F>
aa a aa_ TRANSLATION OF CELD DOM1/2 a_aaaa_,
SEQ I D NO:2
¦ SUBSTITUTE SHEET ~
W O 94/00578 21 3 ~ O 9 9 ~ PC~r/A U93/00307
,_ ~
celD doml/2 Tr~nc]are~ SY~-~n~e
2060 2070 2080 2090 2100
~ ~ ~
GAT GCC GCT GGT AAG AAT GAC CTT GAA TGG AAT ATT AAC TTA ATG AAG AAG AGA
CTA CGG CGA CCA TTC TTA CTG GAA CTT ACC TTA TAA TTG AAT TAC TTC TTC TCT
D A A G K N D L E W N I N L M K X R~
_ a a _ a a _ a _TRANSLATION OF CELD DOMl/2 a a _ a _ a _ a _
2110 2120 2130 2140 2150 2160
TTT GTT GAT CAA GGT ATT CCA ATG ATT CTT GGT GAA TAT GGT GCC ATG AAT CGT
AAA CAA CTA GIT CCA TAA GGT TAC TAA G~A CCA CTT ATA CCA CGG TAC TTA GCA
F V D - Q G I P M I L G E Y G A M N R>
a a a a a _TRANSLATION OF CELD DOMl/2 a a a a _ a
2170 2180 2190 2200 2210
GAT AAT GAA GAA GAT CGT GCA GCT TGG GCT GAA TTC TAC ATG GAA AAG GTC ACT
CTA TTA CTT CTT CTA GCA CGT CGA ACC CGA CTT AAG ATG TAC CTT TTC CAG TGA
D N E E D R A A W A E F Y M E K V T>
a a a a a _TRANSLATION OF CELD DOMl/2 a a a a a _
2220 2230 2240 2250 2260
~ ~ . . .
GCT ATG GGA GTT CCA CAA GTC ~CG TGG GAT AAT GGT ATC TTT GAA GGT ACC GGT
CGA TAC CCT CAA GGT GTT CAG ACC ACC CTA TTA CCA TAG AAA CTT CCA TGG CCA
A M G V p Q V W W D N G I F E G T G>
a a a a a _TRANSLATION OF CELD OOMl/2 a a a _ a _ a _ >
2270 2280 2290 2300 2310 2320
~ . . . ~ ~
GAA CGT TTT GGT CTT CIT GAT CGT AGA AAC TTA AAG ATT GTT TAT CCA ACT ATCCTT GCA AAA CCA G~A GAA CTA GCA TCT TTG AAT TTC TAA CAA ATA GGT TGA TAG
.E R F G L L D R R N L K I V Y P T I>
a a a a a ~ NS1~TION OF CELD DOMl/2 a a a a a
2330 23q0 2350 2360 2370
~ ~ --
GTT GCT GCT TTA CAA AAG GGA AGA GGT TTA GAA GTC AAT GTT GTT CAT GCT GTT
CAA CGA CGA AAT GTT TTC CCT TCT CCA AAT CTT CAG TTA CAA CAA GTA CGA CAA
V A A L Q K G R G L E V N V V H A V~
a. a a _ a a _SRANSLATION OF CELD DOMl/2 a a a a a _ >
2380 2390 2400
~ ~
GAA AAA AAA ACC AGA AGA ACC AAC
CTT TTT TTT TGG TCT TCT TGG TTG
E X K T R R T N>
_ TRANSLATION OF CELD DOMn _ >
SEQ ID NO:2
- ¦1 8U~ 111 iJTE SHEET
WO 94/00578 21 3q 0 99 ~ ` PCI'/AU93/0030'
celD dom3 46
SF~nre Range: 1 to 1437
CCA GAA GAA CCA ACT AAG ACT A GAA CCA GTT GAA CCA ACT GAA ACT ACT AGT
CTT CTT GGT TGA ~1l~ TGA TGA CTT GGT CAA CTT GGT TGA CTT TGA TGA TCA
P E E P T K T T E P V E P T E T T S>
a _ a _ a a _ TRANSLATION OF CELD DOM3 TRUNC_a _ a a _ a a >
100
CCA GAA GAA CCA ACT GAA ACT ACT AAT CCA GAA GAA CCA ACC GGT AAT ATT CGT
GGT CTT CTT GGT TGA CTT TGA TGA TTA GGT CTT CTT GGT TGG CCA TTA TAA GCA
P E E P T E T T N P E E P T G N I R~
_ a _ a a___a TRANSLATION OF CELD DOM3 TRUNC_a a _ a _ a _ a _ >
110 120 130 140 150 160
GAT ATT TCA TCT AAG GAA TTA ATT AAA GAA ATG AAT TTC GGT TGG AAT TTA GGT
CTA TAA AGT AGA TTC CTT AAT TAA TTT CTT TAC TTA AAG CCA ACC TTA AAT CCA
D I S S K E L I K E M N F , G W N L- G>
_ a a a a _ TRU~L~TION OF CELD DOM3 TRUNC_a a a a a _ >
170 180 190 200 210
AAT ACT TTA GAT GCT CAA TGT ATT GAA TAC TTA AAT TAT GAT AAG GAT CAA ACT
TTA TGA AAT CTA CGA G,TT ACA TAA CTT ATG AAT T,TA ATA CTA TTC CTA GTT TGA
N T L D A Q C I E Y L N Y D K D Q T>
a a, a a _ TR~N5LATION OF CELD DOM3 TRUNC_a _ a _ a _ a a _ >
220 230 240 250 260 270
GCT TCT GAA ACT TGC TGG GGT AAT CCA AAG ACT ACT GAA GAT ATG TTC AAG GTT
CGA AGA CTT TGA ACG ACC CCA TTA GGT TTC TGA TGA CTT CTA TAC AAG TTC CAA
A S E T C W G N P K T T E D M F K V>
a a a a, TRANSLATION OF CELD DCM3 TRUNC_a a _ a _ a a _ >
280 290 300 310 320
TTA ATG GAT AAC CAA TTT AAT GTT TTC CGT ATT CCA ACT ACT TGG TCT G~T CAC
AAT TAC CTA TTG GTT AAA TTA CAA AAG GCA TAA GGT TGA TGA ACC AGA CCA GTG
L M D N Q F N V F R I P T T W S G H>
a a a a TRANSLATION OF CELD DOM3 TRUNC_a a a a a _ >
330 3g0 350 360 370
TTC GGT GAA GCT CCA GAT TAC AAG ATT AAT GAA AAA TGG TTA AAG AGA GTT CAT
AAG CCA CTT CGA GGT CTA ATG TTC TAA TTA CTT TTT ACC AAT TTC TCT CAA GTA
F G E A P D Y K I N E K W L K R V H>
a _ a _ a a TRANSLATION OF CELD DOM3 TFUNC_a a _ a a a _ >
380 390 400 410 420 430
GAA ATT GTT GAT TAT CCA TAC AAG AAT GGA GCT TTC GTT ATC TTA AAT CTT CAC
TAA CAA CTA ATA GGT ATG TTC TTA CCT CGA AAG CAA TAG AAT TTA GAA GTG
E I V D Y P Y K N G A F V I L N L H>
a~a~a,_a_~ANSL~TICi~J OF OEID ~M3 TRDNC_a a_a a a
440 450 460 470 480
CAT GAA ACT TGG AAC CAT GCT TTC TCT GAA ACT CTT GAC ACT GCC AAG GAA ATT
GTA CTT TGA ACC TTG GTA CGA AAG AGA CTT TGA GAA CTG TGA CGG TTC CTT TAA
H E T W N H A F S E T L D T A K E I>
a _ a a _ a _ TFU~E~LATION OF CELD DOM3 TRUNC_a a a a a _ >
490 500 510 520 530 5q0
SEQ ID NO: 3
SUB~TITUTE SHEET
WO 94/00~78 213 4 0 ~ 9 PC~r/AU93/00307
4 7
celD dom3
TTA GAA AAG ATT TGG TCT CAA ATT GCT GAA GAA TTT AAG GAT TAT GAT GAA CAC
AAT CTT ~1-l~ TAA ACC AGA GTT TAA CGA CTT CTT AAA TTC CTA ATA CTA CTT GTG
L E R I W S Q I A E E F R D Y D E H>
a _ a _ a _ a TRANSLATION OF CELD DOM3 TRoNC_a a a a a >
550 s60 570 s80 sgo
TTA ATT TTT GAA GGA TTA AAC GAA CCA AGA AAG AAT GAT ACT CCA GTT GAA TGG
AAT TAA AAA CTT CCT AAT TTG CTT GGT TCT TTC TTA CTA TGA GGT CAA CTT ACC
L I F E G L N E P R R N D T P V E W~
_ a _ a _ a _ a, TRANSLATION OF CELD DOM3 TRU,NC_a a a a a >
600 610 620 630 640
ACT GGT GGT GAT CAA GAA GGA TGG GAT GCT GTT AAT GCT ATG AAT GCC GTT TTC
TGA CCA CCA CTA GTT CTT CCT ACC CTA CGA CAA TTA CGA TAC TTA CGG CAA AAG
T G G D Q E G W D A V N A M N A V F>
a a a a TRANSLATION OF ~Fr-n DOM3 TRU~C_a, a a _ a a >
6s0 660 670 680 690 700
TTA AAG ACT ATT C~-l~ AGT TCT GGT AAT AAT CCA AAG CGT CAT CTT ATG ATC
AAT TTC TGA TAA GCA TCA AGA CCA CCA TTA TTA GGT TTC GCA GTA GAA TAC TAG
L R T I R S S G G N N P R R H L M I>
a a _ a_ a _ TRANSLATION OF CELD DOM3 TRUNC_a a a a a
710 720 730 740 7so
CCT CCA TAT GCT GCT GCT TGT AAT GAA AAT TCA TTC AAG AAC TTT ATT TTC CCA
GGA GGT ATA CGA CGA CGA ACA TTA CTT TTA AGT AAG TTC TTG AAA TAA AAG GGT
P P Y A A A C N E N S F R N F I F P~
a a _ a a TRANSLATION OF CELD DOM3 TRUNC_a _ a a _ a a _ >
760 770 780 790 800 810
GAA GAT GAT GAC AAG GTT ATT GCT TCT GTT CAT GCT TAT GCT CCA TAC AAC TTT
. CTT CTA CTA CTG TTC CAA TAA CGA AGA CAA GTA CGA ATA CGA GGT ATG TTG AAA
E D D D R V I A S V H A Y A P Y N F>
_ a a a a TRANSLATION OF ~F~n DOM3 TRUNC_a a _ a _ a _ a >
820 830 840 850 860
GCC TTA AAT AAT GGT GCA GGA GCT GTT GAT AAG TTT GAT GCC GCT GGT AAG AA~
CGG AAT TTA TTA CCA CGT CCT CGA CAA CTA TTC AAA CTA CGG CGA CCA TTC TTT
A L N N G A G A V D R F D A A G K R>
a a a a _ TRANSLATION OF CELD DoM3 TRUNC_a a a a a _ >
870 880 890 900 91o
~ ~ ~
GAT CTT GAA TGG AAC ATT AAC TTA ATG AAG AAG AGA TTT GTT GAT CAA GGT ATT
CTA GAA CTT ACC TTG TAA TTG AAT TAC TTC TTC TCT AAA CAA CTA GTT CCA TAA
D L E W N I N L M R R R F V D Q G I>
_ a a a a _ TRANSLATION OF ~FTn ~OM3 TRUNC_a _ a, a a a _ >
920 930 940' 950 960 970
~ ~ ~
CCA ATG ATT CTT GGT GAA TAT GGT GCC ATG AAC CGT GAT AAT GAA GAA GAA CGT
GGT TAC TAA GAA CCA CTT ATA CCA CGG TAC TTG GCA CTA TTA CTT CTT CTT GCA
P M I L G E Y G A M N R D N E E E '~
a, a a _ a _ TRANSLATION OF CELD DOM3 TRUNC_a a a a a _
980 990 lOoO lolO 1020
~ ~ --
GCT ACA TGG GCT GAA TTC TAC ATG GAA AAG GTC ACT GCT ATG GGA GTT CCA CAA
- CGA TGT ACC CGA CTT AAG ATG TAC CTT TTC CAG TGA CGA TAC CCT CAA GGT GTT
A T W A E F Y M E R V T A M G V P Q>
_ a _ a a a _ TRANSLATION OF CELD DOM3 TRUNC_a a _ a a a
SEQ ID NO:3
~ 5Ut~ JTE~ 8HEET ~
WO 94/00578
PCI`/AU93/0031`"
21~9~
48
celD dom3 P
1030 1040 1050 1060 1070 1080
GTC TGG TGG GAT AAT GGT GTC TTT GAA GGT ACC GGT GAA CGT TTT GGT CTT CTT
CAG ACC ACC CTA TTA CCA CAG A~A CTT CCA TGG CCA CTT GCA AAA CCA GAA GAA
V W W D N G V F E G T G E R F G L L~
_ a a _ a _ a TRANSLATION OF CELD D0M3 TRUNC_a a _ a _ a a _
1090 1100 1110 1120 1130
GAT CGT AAA AAC TTA AAG ATT GTT TAT CCA ACT ATC GTT GCT GCT TTA CAA AAG
CTA GCA TTT TTG AAT ITC TAA CAA ATA GGr TGA TAG CAA CGA CGA AAT GTT TTC
D R X N L X I V Y P T I V A A L Q K~
_ a _ a a _ a _ TRANSLATION OF CELD DOM3 TRUNC_a a a a a
1140 1150 1160 1170 1180
GGA AGA GGT TTA GAA GTT AAG ~1-l GTT CAT GCA AAT GAA GAA GAA ACA GAA GAA
CCT TCT CCA AAT CTT CAA TTC CAA CAA GTA CGT TTA CTT CTT CTT TGT CTT CTT
G R G L E V K V V H A N E E E T E E>
a a _ a _ a _ TRANSLATION O~ CELD DOM3 TRUNC_a a a a a
1190 1200 1210 1220 1230 12~0
TGT TGG ~CT GAA A~G TAT GGT TAT GAA TGT TGT TCT CCT AAC AAT ACT AAG GTT
ACA ACC AGA CTT TTC ATA CCA ATA CTT ACA ACA AGA GGA TTG TTA TGA TTC CAA
C W S E X Y G Y E C C S P N N T K V>
a a a a TRANSLATION OF CELD D0M3 TRUNC_a a a a a _
1250 1260 1270 1280 1290
GTA GTC AGT GAT GAA AGT GGA AAT TGG GGT GTT GAA AAT GGT AAT TGG TGT GGT
CAT CAG TCA CTA CTT TCA CCT TTA ACC CCA CAA CTT TTA CCA TTA ACC ACA CCA
V V S D E S G N W G V E N G N W C G>
a a a a _ TRANSLATION OF CELD DOMB TRUNC_a a a a a _
1300 1310 1320 1330 1340 1350
GTT CTT AAA TAC ACT GAA AAA TGT TGG TCA CTT CCA TTT GGA TAC CCA TGT TGT
CAA GAA 'TTT ATG TGA CTT TTT ACA ACC AGT GAA GGT AAA CCT ATG GGT ACA ACA
V L K Y T E K C W S L P F G Y P C C>
_ a a a _ a TRANSLATIoN OF CELD DOM3 TRUNC_a _ a a _ a a _
1360 1370 1380 1390 lg00
CCA CAT TGT AAG GCT CTT ACT AAG GAT GAA AAT GGT AAA TGG GGA GAA GTA AAT
GGT GTA ACA TTC CGA GAA TGA TTC CTA CTT TTA CCA TTT ACC CCT CTT CAT TTA
P H C X A L T K D E N G K W G E V N>
a a a _ a _ TRANSLATION OF CELD DoM3 TRUNC_a a _ a a a _
1410 1420 1430
GGT GAA TGG TGT GGT ATT GTT GCT GAT AAA TGT
CCA CTT ACC ACA CCA TAA CAA CGA CTA TTT ACA
G E W C G I V A D X C>
_ a _ TRANSLATION OF CELD DOM3 TRUNC _ a _ >
SF.Q ID NO: 3
3 ~3UE~8~ 1TE SHEET
W094/00578 ~1 3q ~q~ pcr/Aug3/oo3o7
-
49
ATGGCTAGC AATGG~AAAAAG~
M A S ~ G X ~
TTACTGTCGGTAATGGACAAAACCAACATAAGGGTGTCA~CGATGGT~TCAGTTATGAAA
F ~ v G ~ G Q ~ Q ~ K G V N D G F S Y E
TCTGGTTAGATAACACTGGTG~TAACGGTTCTATGACTCTCGGTAGTGGTGCAACTTTCA
I W L D N T G G ~ G S M T L G S G A T F
AGGCTGAATGGAATGCAGCTGTTAACCGTGGTAACTTCCrTGCCCGTCGTGGTCTTGACT
K A E U N A A V N ~ G N F L A R ~ G ~ D
TCGGTTCTCAAAA~AAGGCAACCGATTACGAC~ACATTGGATTAGATTATGCTGCTACTT
F G S ~ K K A T D Y D ~ I G L D Y A
ACAAACAAACTGCCAGTGCAAGTGGTAACTCCCGTCTCTGTGTATACGGATGGTTCCAA~
Y K Q T A S A S G ~ S ~ L C V Y G W F Q
ACCGTGGACTTAATGGGGTTCCTTTAGTAGAATACTACAT~ATTGAAGATTGGGTTGACT
N R G L N G V P ~ V E Y Y I I E D W V D
GGGTTCCAGATGCACAAGGAAAAATG~TAACCATTGATGGAGCTCAATATAAGATTTTCC
W V ~ D A Q G R M v T I D G A Q Y ~ I F
AAATGGATcAcAcTGGTccAAcTATcAATGGTGGTAGTGAAAccTTTAAG~AATAcTTcA
~ M D ~ T G P T I N G G S E T F K Q Y
GTGTCCÇTCAACAAAAGAGAACTTCTGGTCATATTACTGTCTCAGATCACTTTAAGGAAT
S v R Q Q ~ ~ T S G ~ I T V ~ D ~ F ~ E
GGGCCAAACAAGGTTGGGGTATTGGTAACCTT~A~GAAGTTGCTTTGAACGCCGAAG~TT
~ A ~ Q G W G I G N L Y E V A ~ ~ A E G
G~CAAAGTAGTGGTGTTGCTGATGTCACCTTATTAGATGTTTACACAACTCCAAAGGGTT
W Q S S G V A D Y T L L D V Y T T P K G
CTAGTCCAGCC~CCTCTGCCGC~CCTCGT TM
S S P A T S A A P R
SEQ ID NO:4
SUBSTITUTE SHEET
