Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR THE STABILIZATION OF PROTEINS AND THE
THERMOSTABILIZED ALCOHOL DEHYDROGENASES PRODUCED THEREBY
TECHNICAL FIELD OF THE INVENTION
The present invention generally relates to a method
for the directed evolution of proteins. In particular,
the method is directed to stabilization of proteins such
as dehydrogenases, and particularly is directed to a
method for improving the thermostability of
dehydrogenases such as alcohol dehydrogenases. The
present invention also relates to thermostabilized
alcohol dehydrogenases produced according to this method.
BACKGROUND OF THE INVENTION
Biocatalysts are enzymes which can specifically and
efficiently expedite chemical reactions such as the
synthesis of chemical compounds and biopolymers (Dixon et
al., Enzymes (Academic Press, New York: 1979)).
Biocatalysts are the key players in a number of important
industrial synthetic and degradative applications
including, but not limited to, the following:
~ Synthetic Applications - Biocatalysts currently are
employed as feasible alternatives to traditional
catalysts, especially for the synthesis of chiral
intermediates, or in the reduction of the number of
protection/deprotection steps.
~ Biodegradation Applications - Biocatalysts currently
are employed as enzymatic degradation agents for
environmental pollutants such as PCBs, chlorinated
hydrocarbons, RDX, halogenated organic compounds,
TNT, and other byproducts of industrial production
that present significant health risks.
~ Diagnostics and Biosensors - Biocatalysts currently
are employed as detection agents in diagnostic tests
and as biosensors which require enzyme durability.
~ Other large-scale industrial applications -
Biocatalysts currently are employed as catalysts in
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the production of fuel supplies through conversion
of agricultural feedstocks.
One enzyme that is of considerable utility in
current enzymatic processes is the dehydrogenase. In
particular, alcohol dehydrogenases are enzymes that
command formal, reversible, two-electron chemistry in
which alcohols are oxidized to the corresponding ketones.
Depending on the precise reaction conditions, ketones can
be reduced to the respective alcohols via a
stereospecific delivery of a hydride equivalent catalyzed
by the enzyme coupled to a bound cofactor such as NADH or
NADPH (Lemiere, "Alcohol Dehydrogenase Catalyzed
Oxidoreduction Reactions in Organic Chemistry", _In
Enzymes as Catalysts in Or anic Synthesis, Schneider et
al., Eds. (1986) p. 17). This system thus provides a
mild, extremely sensitive route to chiral compounds,
without contamination from undesired, competing
reactions.
Such chiral compounds can be used, especially by the
pharmaceutical industry, for the preparation of chiral
therapeutics, and for effectively generating a wide
variety of compounds having the capacity for industrial
scale-up (Seebach et al., Org. Synth., 63, 1- (1984);
Bradshaw et al., J. Org. Chem., 57, 1532(1992); Hummel,
Biotechnol. Lett., 12, 403(1990)). In particular,
dehydrogenases show promise for commercial application in
the preparation of unusual amino acids and ~3-
hydroxyketones, and in the resolution of racemic alcohols
(Benoiton et al., J. Am. Chem. Soc., 79, 6192 (1957);
Casy et al., Tetrahedron Lett., 33, 817 (1992); Jacovac
et al., J. Am. Chem. Soc., 104, 4659-4665 (1982); Jones
et al. Can. J. Chem., 60, 19 (1982)). Of the
dehydrogenases, horse liver alcohol dehydrogenase (HLADH)
is one of the most commonly used.
For an enzyme biocatalyst such as HLADH to prove
useful in a wide-scale, practical, industrial
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application, it is important that the biocatalyst possess
the ability to survive harsh, dynamic, environmental and
handling conditions inherent to large-scale commercial
processes. These conditions include nonrefrigerated
storage, and exposure to organic cosolvents and high
reaction temperatures, as well as more idiosyncratic
demands imposed by a particular industrial application.
To date, one of the greatest challenges associated
with biocatalyst implementa~.ion is that of overcoming an
overall intrinsic instability that results in a
requirement for special preparative approaches and
handling conditions. Many methods have been used in an
attempt to stabilize certain proteins. Rational protein
engineering has allowed the redesign of proteins with
altered properties such as enhanced stability, shifted pH
optima, and different substrate specificities (see, e.g.,
Bryan et al., Proteins, 1, 326-334 (1986); Pantoliano et
al., Biochemistry, 26, 2077-82 (1987); Carter et al.,
Science, 237, 394-399 (1987); Wells et al., "Designing
substrate specificity by protein engineering of
electrostatic interactions", , 84,1219-1223(1987);
Grutter et al., Nature, 277, 667-669 (1979)).
While potentially an extremely powerful tool,
rational protein engineering can be extremely time-
consuming and expensive, and currently can be employed
only for a very small number of enzymes having well-
defined crystal or solution structures. Moreover, since
the approach is tailored to a specific enzyme, it
typically cannot be generalized to other enzyme species.
Other post-production stabilization methods such as
immobilization (Macaskie et al., FEMS Microbiol Rev.,
14,351-67 (1994); Shtelzer et al., Biotechnol. Appl.
Biochem., 15, 227-35 (1992); Phadke, Biosystems, 27, 203-
6 (1992)), or use of cross-linked enzymes (Navia et al.,
"Crosslinked enzyme crystals as robust biocatalysts",
Proceedings of the Materials Research Society 1993
Symposium, Biomolecular Materials by Design (1993)),
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suffer some of the same as well as further shortcomings,
and similarly, are often too expensive to implement.
By contrast, directed evolution potentially can
provide a practical approach to tailoring enzymes for a
wide range of applications (Shao et al., "Engineering New
Functions and Altering Existing Functions", Current
Opinion in Structural Biology, in press (1996)). In
support of this, enzymes have been shown to be highly
adaptable molecules over evolutionary time scales. Many
enzymes catalyzing very different reactions appear to
have come about by divergent evolution, acquiring diverse
capabilities by the processes of random mutation,
recombination, and natural selection.
Thus, there remains a need for an effective means to
randomly engineer better enzymes, particularly
dehydrogenases, and especially, HLADH. The present
invention seeks to overcome some of the aforesaid
problems of enzyme design. In particular, it is an
object of the present invention to provide a method for
the directed evolution of enzymes, particularly
dehydrogenases, and especially HLADH. It further is an
object of the present invention to provide a method for
stabilizing, e.g. improving the thermostability of
enzymes such as dehydrogenases. Such a method of
stabilizing dehydrogenases (particularly HLADH) would
present a major advancement in the field since it would
extend the shelf life, longevity, and active temperature
range of these enzymes. These and other objects and
advantages of the present invention, as well as further
inventive features, will be apparent from the description
of the invention provided herein.
BRIEF SUMMARY OF THE INVENTION
Briefly, the present invention provides, inter alia,
a method for the stabilization of a protein (particularly
for the stabilization of an alcohol dehydrogenase such as
horse liver alcohol dehydrogenase (HLADH), general
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enrichment/selection means that can be employed in
Escherichia and Thermus to select for cells having
altered levels of alcohol dehydrogenase activity as
compared to a wild-type cell, thermostabilized HLADH
5 proteins and nucleic acid sequences encoding same, as
well as plasmids and hosts cells comprising the nucleic
acid sequences.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a diagram that generally depicts the
approach of the present invention for the accelerated
evolution of enzymes. A pool of mutants of the
particular gene is obtained by means such as spontaneous,
directed, chemical, or PCR-mediated mutagenesis. The
mutants of interest (i.e., having the particular
stabilized feature) are identified by means of a screen
or selection (A), and optionally, compatible mutations
can be combined (e. g., by gene splicing, in vitro
recombination, and the like) to enhance the stability
even further (B).
Figure 2 is a digitized image of results of a
filter assay for alcohol dehydrogenase activity which
demonstrates that wild-type HLADH is rapidly inactivated
at 75°C: no heat treatment (A); 5 minutes of heat
treatment at 75°C (B); 10 minutes of heat treatment at
75°C (C); 15 minutes of heat treatment at 75°C (D); 20
minutes of heat treatment at 75°C (E); and 50 minutes of
heat treatment at 75°C (F).
Figure 3 is a partial restriction map of the
plasmid pTG450 which contains the adh gene from plasmid
pBPP cloned into a pTG100kan'=z Thermus shuttle vector.
Figure 4 is a bar chart that depicts the increased
thermostability of HLADH mutants produced according to
the invention at 70°C. Cells containing pGEM-T (i.e.,
having no HLADH gene) did not show any HLADH activity.
Figure 5 is the sequence of adh gene [SEQ ID NO:1]
that encodes the HLADH protein [SEQ ID N0:2], with the
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location of certain mutations produced according to the
invention identified as the boxed regions.
DETAILED DESCRIPTION OF THE INVENTION
$ The present invention provides, among other things,
a method for stabilizing a certain feature of a protein
(e.g., stability at a certain temperature, stability in
the presence of certain reagents, etc.). In particular,
the method of the invention provides a method for
thermostabilizing a protein. Namely, the invention
preferably provides a method of obtaining nonnative
protein having a thermostability that is increased over
that of the native version of said protein, as further
described herein.
According to the invention, a "native" protein is
the protein as it generally is found in nature. By
contrast, a "nonnative" protein differs from the native
protein in that it has been modified by human
intervention, i.e., at either the level of the protein
or its encoding DNA (e.g., by recombinant means to
directly alter the genome; by unique selection and
forced mutation; by random mutagenesis). Moreover, a
"protein" desirably can be either an entire protein, or
a portion of a protein (e. g., as where a chimeric
nonnative protein results from either transcriptional or
translational gene fusion). Similarly, a "nonnative
protein" in some applications (e.g., applications for
further study) may be a peptide (i.e., an incomplete
protein), as where the peptide is chemically synthesized
or, where a gene's coding sequence is transcribed or
translated in vitro or, is produced by chemical
processing of a complete protein.
A preferred protein for stabilization, particularly
thermostabilization according to the invention is a
dehydrogenase, particularly an alcohol dehydrogenase,
and especially horse liver alcohol dehydrogenase (e. g.,
as obtained from plasmid pBPP, and/or as set forth in
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SEQ ID N0:2). Notably, with respect to SEQ ID N0:2, this
protein does not initiate with methionine (Met).
However, other varients of horse liver alcohol
dehydrogenase produced by in vitro synthetic reactions,
by means of chemical synthesis or, in other hosts (e. g.,
an eukaryotic host or other prokaryotic host cell) may
possess a methionine residue in the first position of
the protein. The numbering of residues in such proteins
of course, would differ somewhat from that of SEQ ID
N0:2. Namely, the second position of the aforementioned
protein would be equivalent to the first position of the
protein of SEQ ID N0:2. Of course, the ordinarily
skilled artisan would know how to compare equivalent
regions of proteins.
Desirably, other proteins (particularly proteins
having capacity for industrial implementation) can be
stabilized (e.g., thermostabilized) according to the
invention. For instance, an alcohol dehydrogenase
protein can be employed from another species. It is
anticipated that this approach can be employed with
alcohol dehydrogenases from other species based on the
similarities between certain of the various alcohol
dehydrogenases. Also, a protein according to the
invention optionally can be another type of
dehydrogenase, e.g., another type of NAD+(P)-linked
dehydrogenase including, but not limited to, malate
dehydrogenase, lactate dehydrogenase, isocitrate
dehydrogenase (NADP+), hydroxylacyl CoA dehydrogenase,
glyceraldehyde 3-phosphate dehydrogenase, and glucose 6-
phosphate dehydrogenase (NADP+).
In a preferred embodiment, the method can be
employed to thermostabilize a horse liver alcohol
dehydrogenase. This method generally is depicted in
Figure 1. Preferably the method comprises:
(a) obtaining in a vector a gene that encodes the
native protein;
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(b) mutating the vector at more than one position
in the gene to produce a vector library of cells
comprising mutated versions of the gene;
(c) introducing the vector library en masse into
cells of a strain in which the majority of the mutated
versions of the gene are transcribed and translated to
produce a cell library;
(d) screening the cell library to identify a cell
comprising a mutated version of the gene that encodes a
nonnative protein having a thermostability that is
increased over that of the wild-type verson of the
protein; and
(e) purifying the cell from the cell library.
According to the invention, "gene that encodes said
protein" can comprise a recombinant or nonrecombinant
sequence, i.e., a sequence that is present as found in
nature (i.e., encodes a native amino acid sequence) or,
has been modified, for instance by the introduction of
mutations (e. g., point mutations, insertions, deletions,
or rearrangements) to comprise a nonnative amino acid
sequence or, can be a mixture of native and nonnative
amino acid sequences. Similarly, a recombinant gene may
conjoin coding sequences (either in entirety or in part)
with regulatory sequences (e. g., transcription
initiation, transcription termination, translational
start or stop sites, protein secretion sequences, and
the like) which are not typically conjoined in nature.
This can allow the production of a protein in a host in
which it normally is not produced (e.g., production of a
eukaryotic protein in a prokaryotic cell). Preferably,
however, the recombinant gene (which can derive, in
entirety or part, from any prokaryotic, eukaryotic,
bacteriophage, or viral source) is capable of being
transcribed and translated in a prokaryotic cell,
particularly, a cell comprising a member of the genuses
Escherichi or Thermos.
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Thus, preferably a host cell in the context of the
present invention (i.e., which can be employed in a
method of stabilizing proteins) is a member of the
kingdom Bacteria, Archaea, or Eukarya. In particular,
S preferably a cell employed in the method of stabilizing
(particularly thermostabilizing) proteins according to
the invention is a thermophile or hyperthermophile. In
particular, preferably a cell is a member of the genus
Thermos, and desirably is of the species Thermos flavus,
Thermos aqua ticus, Thermos thermophilus, or Thermos sp.
Optimally a cell is either an Escherichia coli cell or a
Thermos aquaticus cell.
The vector in which the gene of interest is
subcloned can be any vector appropriate for delivery of
a gene to a cell. For instance, the vector can be a
plasmid, bacteriophage, virus, phagemid, cointegrate of
one or more vector species, etc. Optimally, however, a
vector is one that can be employed for gene expression
in a prokaryotic cell such as a Thermos or Eshcerichia
cell. It also is preferable that a vector have an
ability to shuttle between different cells, e.g.,
between a Thermos and an Eschericia cell. One such
vector that can be employed in the context of the
invention is the vector pTG450.
The preferred method of the invention calls for
mutating a vector containing the gene encoding the
protein to be stabilized. Any method of mutagenesis such
as is known to those skilled in the art and particularly
as is described in the following Examples, can be
employed in the method of the invention for generating a
mutated gene. Desirably a PCR-based (error prone)
approach, especially as set out as follows, is employed
for mutagenesis. However, other mutagens (e. g., chemical
mutagens such as hydroxylamine), also can be employed.
In the preferred method of mutagenesis employed in
the invention, desirably the vector is mutated at more
than one position in the gene of interest. This can be
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assessed by means known in the art and as described in
the Examples. Such mutagenesis in more than one position
in the gene will result in a "vector library" comprising
mutated versions of a gene, particularly of a horse
5 liver alcohol dehydrogenase gene, which are present in
the library mixture.
The vector library can be introduced en masse into
cells (e.g., by transformation). Since the vectors and
the cells employed for these methods are selected to be
10 compatible, and the gene is engineered (e.g., as
described below) to contain or to be flanked by any
sequences necessary for its expression, it is expected
that such introduction will result in the transcription
and ensuing translation of the introduced gene.
Moreover, such en masse introduction will result in the
generation of a cell library comprising a mixture of
cells transformed with plasmids having differing mutated
genes. In some instances, it may be desirable to
reisolate the vectors from the cell library (e.g., by a
plasmid isolation or other vector isolation protocol),
excise out the mutated gene, and subclone the mutated
gene into another vector (e.g., a vector that has not
been mutagenized).
Following the generation of the cell library, the
cells preferably are screened under conditions that
allow identification of a cell comprising a mutated
version of the gene of interest that encodes a nonnative
protein having a protein that is stabilized (e. g.,
thermostabilized) over that of the wild-type (i.e.,
native) versions of the protein. A variety of selection
means can be employed in accordance with the method of
the present invention and, in particular, the selection
means identified in the Examples which follow can be
employed. Of course, one of ordinary skill in the art
could modify these methods such that they are adapted
for a particular host cell and/or a particular protein
of interest. Desirably, however, screening conditions
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are employed that provide for enrichment and/or
selection for a cell containing nonnative DNA that
encodes a protein having a particular feature of
interest.
In particular, when the protein being stabilized
according to the invention is an alcohol dehydrogenase,
and particularly HLADH, the screen preferably can be
carried out at increased temperature. For instance,
desirably, screening is done at temperature a few
degrees above and a few degrees below the temperature at
which the native (i.e., wild-type) alcohol dehydrogenase
is inactivated in the particular host cell employed for
screening.
According to this invention, "increasing the
thermostability" of a nonnative protein means: (a)
increasing the length of time at which a nonnative
protein exhibits activity as compared to the wild-type
protein; (b) increasing the temperature at which a
nonnative protein exhibits activity as compared to a
wild-type protein; or (c) increasing the length of time
and temperature at which a nonnative protein exhibits
activity as compared to a wild-type protein. A protein's
activity can be determined by a variety of tests that
differ with the various proteins to be tested. A few
representative tests that can be employed in the method
of the invention are set out in the following Examples.
Preferably, however, "activity" means a detectable
activity ranging from 10 to 90 units. For instance,
whereas a wild-type protein might exhibit 10% activity
at a defined temperature for a set amount of time, a
thermostabilized enzyme might exhibit 10% activity at
the same temperature for an increased amount of time,
and/or might exhibit an activity at an increased
temperature at which the native protein exhibits reduced
or no activity.
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The screening methods also desirably can be done,
for instance, in the presence of alcohol, optionally at
a lowered pH.
Following screening of cells to identify those
having the desired traits) imparted by the mutated
gene, optionally, cells exhibiting the trait can be
further isolated. Vectors containing mutated versions of
the gene of interest optionally can be further
mutagenized by repeating steps (b) through (e) above to
further stabilize the encoded protein.
The present invention accordingly also provides
screens that can be employed to select for or against
cells having altered ADH activity. For instance, the
invention provides a method for selecting against growth
of Eschericia coli recombinant cells which comprise
levels of alcohol dehydrogenase that are higher than
those of wild-type Eschericia coli cells. According to
this invention, "growth" means an increase in cell mass,
or some other evidence of cell metabolism such as one of
ordinary skill in the art knows how to detect, or is
described in the following Examples. An "absence of
growth" means growth is not measurable by common
procedures (e. g., visual or spectrophotometric
observation and the like) or, cell killing. Cell killing
can be determined by any well known means, e.g., visual
observation, release of cell components, vital staining
etc.
Thus the E.coli selection method comprises growing
said recombinant cells under conditions selected from
the group consisting of, wherein ethanol is present in a
concentration of about 10%, isopropanol is present in a
concentration of about 40, and propanol is present in a
concentration of about 2%, with the proviso that the
wild-type cells exhibit reduced or an absence of growth
under these conditions.
The present invention similarly provides a method
for selecting for growth of Thermus flavus recombinant
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cells which comprise levels of alcohol dehydrogenase
that are higher than those of wild-type Thermus flaws
cells. This method comprises growing the recombinant
cells under conditions selected from the group
consisting of wherein ethanol is present at a
concentration of aboutlo in a liquid or solid medium at
a pH of about 7.0, with the proviso that the wild-type
cells exhibit reduced or an absence of growth under
these conditions.
As mentioned previously, these methods have been
employed to thermostabilize HLADH. In particular, the
invention provides an isolated and purified
thermostabilized HLADH protein comprising a sequence
selected from the group consisting of SEQ ID N0:4, SEQ
ID N0:6, SEQ ID N0:8, SEQ ID NO:10, SEQ ID N0:12, SEQ ID
N0:14, SEQ ID N0:16, SEQ ID N0:18 and SEQ ID N0:20. The
invention also provides genes encoding such protein,
e.g., an isolated and purified nucleic acid comprising a
sequence selected from the group consisting of SEQ ID
N0:3; SEQ ID N0:5, SEQ ID N0:7, SEQ ID N0:9, SEQ ID
NO:11, SEQ ID N0:13, SEQ ID NO:15, SEQ ID N0:17 and SEQ
ID N0:19.
Moreover, the invention provides for plasmids
encoding for such proteins: e.g., a plasmid comprising
one of the aforementioned nucleic acid sequences; and a
plasmid selected from the group consisting of pAD7;
pAD$, pADlO, pAD9l, pAD92, pAD93, pAD95, pADlll, pAD113,
and pTG450.
The invention further preferably provides a method
of increasing the thermostability of horse liver alcohol
dehydrogenase. This method comprises introducing into a
gene which encodes the alcohol dehydrogenase a mutation
at a codon which codes for an amino acid residue at a
position selected from the group consisting of the amino
acid positions, 75, 94, 110, 177, 257, 268, 282, 292,
and 297.
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Examination of the three-dimensional structure of
the HLADH protein will elucidate the manner in which
further amino acid substitutions thermostabilizing the
enzyme can be made, for instance, like-for-like (e. g.,
with acidic amino acids (i.e., aspartic acid, glutamic
acid) being substituted for acidic amino acids; basic
amino acids (i.e., lysine, arginine, histidine) being
substituted for basic amino acids; sulfur containing
amino acids (i.e., cysteine) being substituted for
sulfur containing amino acids; amides (i.e., asparagine,
glutamine) being substituted for amides, aliphatic
nonpolar amino acids (i.e., glycine, alanine, valine,
leucine, isoleucine) being substituted for aliphatic
nonpolar amino acids; and alcoholic, aliphatic, and
aromatic amino acids (i.e., serine, threonine,
thyrosine, phenylalanine, and tryptophan) being
substituted for alcoholic, aliphatic, and aromatic amino
acids.
Additional uses and benefits of the invention will
be apparent to one of ordinary skill in the art.
EXAMPLES
The following examples further illustrate the
present invention but, of course, should not be construed
as in any way limiting its scope.
EXAMPLE 1: Quantitative assay for ADH
in cell extracts.
This example describes a method for the
quantification of ADH in cell extracts, particularly for
the quantitation of HLADH, that can be used according to
the invention.
For this assay, overnight cultures of cells to be
assayed are grown in rich media. The cells are washed,
resuspended in 600 ~l of assay buffer (83 mM KH2P04 [pH
7.31, 40 mM KCl, 0.25 mM EDTA), and sonicated. The assay
~ll~-~T~TtIT~ ~~T ~yJt~~ 2~~
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IS
mixture contains 500 ~.l of cell extract, 100 ~1 EtOH, 20
~.1 100 mM NAD, 830 ~1 buffer and is carried out at room
temperature. The reaction is run for 3 minutes and
absorbence at 340 nM is measured. Using this approach it
is possible to identity a high IPTG inducible activity in
the strains with the HLADH coding sequence under the
control of the lacZ promoter. This method thus produces
a reliable quantitative determination of HLADH activity
present in the cell.
EXAMPLE 2: p-Rosanaline/alcohol plate
screen in E. cola.
This example describes a plate screen for ADH
activity that can be employed, for instance, in E. cola.
p-Rosaniline indicator plates are prepared according
to Conway et al. (Conway et al., 169, 2591-2597 (1987))
by adding 8 ml of p-rosaniline (2.5 mg/ml in 96% ethanol)
and 100 mg of sodium bisulfate to 400 ml batches of
precooled (45oC) Luria agar. Most of the dye is
immediately converted to the leuco form by reaction with
bisulfate to produce a rope-colored medium. Ethanol
diffuses into the E. cola cells to produce the
acetaldehyde by alcohol dehydrogenase. The leuco dye
serves as a sink, reacting with the acetaldehyde to form
a Schiff base which is intensely red. Thus, the plates
can be streaked with a strain or, a strain can be applied
in patches to the plate. Colonies will appear a deeper
intensity of red dependent upon the level of ADH present
in the cell. In particular, by plating appropriate
controls on each plate, it is relatively easy to visually
discern a strain which has a high level of dehydrogenase
(deep red staining), an intermediate level of
dehydrogenase (more moderate red staining), and no
activity (little or no red staining).
This method thus provides a plate screen that can be
employed in the method of the invention.
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EXAMPLE 3: Filter screen for HLADH activity.
This example describes a sensitive plate assay of
ADH activity which also allows colonies to be tested
under different treatment conditions.
This assay relies for manipulation of bacterial
colonies on the binding of the colonies to a
nitrocellulose filter. The assay is carried out by a
modified protocol described by Rellos et al. (Rellos et
al., Protein Expression and Purification, _5, 270-277
(1994)). Namely, a series of temperatures between 65 and
85°C in 5°C increments with incubation times varying from
10 minutes to one hour is analyzed in an attempt to
determine the cutoff of the stability of the HLADH
protein. For these experiments, the source of the adh
IS gene encoding the HLADH enzyme was plasmid pBPP (Park et
al., J. Biol. Chem., 266, 13296-13302 (1991)).
E. coli DHS~, cells containing plasmid pBPP (i.e.,
HLADH') or plasmid pCRII ( i . a . , HLADH ) ( InVitrogen;
Carlsbad, CA) were grown on rich media plates at cell
densities up to about 1,000 colonies per plate and
transferred onto a nitrocellulose membrane. The adhered
cells were lysed in Buffer 1 (10 mM KMes, pH 6.5, 0.5 mM
CoCl2, 0.1% Triton X-100, 50 ~Cg/ml lysozyme, 10 ~Cg/ml
DNAse) in a chloroform bath for about one hour, washed
once in Buffer 2 (10 mM KMes, 0.5 mM CoCl2, 0.2% BSA),
and then washed two more times in Buffer 3 (Buffer 2
without BSA). The filters were then incubated at high
temperatures in Buffer 4 (10 mM glycine, 0.5 mM CoCl2)
and, after washing in Buffer 3, were incubated in the
enzyme-detecting solution (30 mM Tris, pH 8.3, 2%
ethanol, 1 mM NAD+, 0.1 mg/ml phenazine methosulfate, 1
mg/ml nitroblue tetrazolium) at room temperature for 3-5
minutes.
Results of these experiments are depicted in Figure
2. As can be seen in this figure, the experiments
confirm that a 15-20 minute treatment of the filters at
75oC resulted in roughly 90% inactivation of the HLADH
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protein as estimated by the color changes. This
information on the activity of the native protein can be
used as a baseline for the identification and isolation
of mutagenized candidates having altered ADH activity
according to the invention.
EXAMPLE 4: Shuttle vectors and use of a p-rosaniline
assay for verification of the activity
of the HLADH gene in Thermus
In order to allow expression of the HLADH gene in
both Thermus and E. coli, the gene was subcloned into the
Thermus shuttle vector, pTGl00kantr2 to create plasmid
pTG450 depicted in Figure 3. In this construct, the gene
is placed upstream of the thermostable kanamycin
resistance gene (kantr2) which is commanded by the lac
promoter in E. coli, and the leu promoter in Thermus.
An E. coli strain harboring pTG450 has three times
more HLADH activity in the presence of IPTG than the
strain harboring the original pBPP plasmid. When
transformed into Thermus, the adh gene integrates into
the leuB site in the Thermus chromosome by a double
recombination event. For these experiments, Thermus
flavus was transformed with both the HLADH plasmid
pTG100kantr2 (i.e., creating strain TGF353) and the HLADH'
plasmid TG450 (i.e., creating strain TGF650).
The presence of the adh gene in TGF650 was confirmed
by PCR, and both TGF353 and TGF650 cells were assayed
using a variation of the p-rosaniline plate assay
described in Example 2. Namely, the agar overlay
contained the same ingredients described, except TT media
(Weber et al., Bio/Technology, 13, 271-275 (1995); Oshima
et al., International Journal of Systematic Bacteriology,
24, 102-112 (1974)) was employed instead of Luria broth.
A standard p-rosaniline plate can not be used since the
indicator dye will spontaneously convert to the Schiff
base if incubated overnight in the plate as part of this
assay.
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Using this approach, HLADH activity was observed in
the pTG450 Thermus transformants at a level well above
background levels observed for the pTG100kantr2 Thermus
transformants. The activity was observed up to 70°C.
These results thus confirm that a p-rosaniline plate
assay similarly can be employed in the context of the
present invention for screening in Thermus for mutants
having altered ADH activity.
EXAMPLE 5: Development of a Method of HLADH
Selection/Enrichment in E. coli
This example describes a method of negative
selection for growth of E. co~i strains harboring the
adh gene.
For these experiments, E. coli DH5a cells
containing either pTG100kan'r2 (i.e., HLADH-) or pTG450
(i.e., HLADH+) were grown on LB plates with different
alcohols in concentrations ranging from 2% to 120. The
results of one such experiment are displayed in Table 1.
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As can be seen from Table l, E. coli cells
harboring high activity of HLADH (i.e., transformed with
the HLADH'plasmid pTG450) are more sensitive to the
5 presence of the alcohols in high concentrations. This
probably is due to the accumulation of toxic aldehyde
levels in the cells which result from the alcohol
dehydrogenase reaction. Three other alcohols were
tested (i.e., benzyl alcohol, hexyl alcohol, and hexyl
10 amine), but did not give clear results because of their
poor solubility in the media.
The experiment was repeated several times and the
alcohol levels were refined to determine a range
resulting in a clear selection. Three of the alcohols,
15 i.e., ethanol at a concentration of 100, isopropanol at
a concentration of 4%, and propanol at a concentration
of 2%, resulted in clean, negative selection for growth
of E. coli harboring the adh gene.
These results thus confirm that the selection
20 scheme can be employed for the isolation of mutants with
altered ADH activity and, in particular, to select
against E. coli strains having high levels of ADH. Such
a system of negative selection also can be employed to
affirmatively identify mutants having high levels of
ADH. For instance, cells can be replica plated onto a
series of plates from a single master plate prior to
their transfer to nitrocellulose membranes. One of the
plates can be retained, instead of being transferred to
nitrocellulose, and matched against the sensitive cells
identified in the assay. Cells of interest can then be
recovered from the untreated plates.
EXAMPLE 6: Development of a Method of HLADH
Selection/Enrichment in Thermos
This example describes the growth of Thermos
strains in the presence of the high concentrations of
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alcohols as a general method for selecting for growth of
Thermus strains having high levels of ADH activity.
A series of experiments was conducted to develop a
selection using alcohol levels in Thermus. In these
experiments, Thermus flavus strains TGF353 (HLADH-) and
TGF670 (HLADH+) were employed. Each strain was grown
for two days on Thermus rich media (e.g., TT media, as
described in Oshima et al., International Journal of
Systematic Bacteriology, 24, 102-112 (1974)) present in
plates or, was grown overnight in 4 ml of liquid TT
medium, in order to ensure the cells were at the same
physiological stage prior to testing. The test itself
was performed on TT media and Thermus minimal media (Yeh
et al., J. Biol. Chem., 251, 3134-3139 (1976) containing
Casaminoacids (TMIN, CAA). Over a series of many
experiments, the strains were grown on agar plates or in
liquid medium containing various concentrations of
ethanol (i.e., 0.5, l, 2, 4, 6, or 8%), various
concentrations of methanol (i.e., 2, 4, 6, or 8%),
various concentrations of isopropanol (i.e., 0.5, 1, 2,
4 or 6%), various concentrations of propanol (i.e., 1,
2, 4, or 6%), or various concentrations of propanediol
(i.e. 0.5 or 1%). Such experiments further were done at
different pHs, i.e., at pH 7.0, 7.5 and 8.0, for the
various alcohols at different concentrations. The
results of one of these experiments is set out in Table
2.
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As can be seen from this experiment, the HLADH+
strain TGF670 demonstrates higher resistance to alcohols
than the HLADH- strain TGF353. Moreover, this selection
appears to be dependent on pH, with the selection
functioning better at lower pH, especially with ethanol.
The selection thus may work by lowering the pH of the
media-Thermus prefers higher pH for growth, in the range
of pH 7.5-8.5 -- although not enough Thermus
biochemistry is known to make this conclusive.
A similar effect can also be achieved on plates.
However, the primary effect of the screen in Thermus is
to retard growth of cells without the adh gene, not to
completely eliminate it. This also is the case with the
liquid media, indicating that a completely clean
selection in Thermus without background is difficult to
achieve. Nevertheless, this selection means provides a
powerful enrichment, especially in liquid, by selecting
for faster growing cells under the conditions defined.
The results thus confirm that the
enrichment/selection means outlined above can be
employed with Thermus.
EXAMPLE 7: Hydroxylamine mutagenesis of the adh gene.
This example describes mutagenesis of the adh gene
as a representative alcohol dehydrogenase gene using the
mutagen hydroxylamine (HA).
For HA mutagenesis of the adh gene, plasmids pBPP
and pTG450, both of which contain this gene, were treated
with HA using a standard approach. Namely, approximately
8 ~.g of plasmid DNA was mixed with 0.5 M NHZOH and
incubated at 37°C for various lengths of time. For
example, aliquots were taken at 1, 2, 3, or 4 hours
following treatment, or following overnight exposure to
the mutagen. The plasmid DNA was then transformed into
E. coli strain DH5a, and plated onto LBAPioo Plates ( i . a . LB
plates containing 100 ~g/ml ampicillin). Transformants
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were analyzed by the ADH filter assay described in
Example 3, and also using the p-rosaniline assay
described in Example 2 to estimate the efficiency of
mutagenesis.
After overnight treatment, only 3 - 4% plasmids
treated with HA remained active. Plasmids treated by HA
under conditions providing ~50% of inactivation of the
adh were then transformed into E. coli strain NM554
(obtained from New England Biolabs) to obtain 500 - 700
transformant colonies per plate. These colonies were
analyzed by the nitrocellulose filter ADH assay described
in Example 3. For heat inactivation'of ADH, the filters
were incubated for 15 minutes at 70 °C in a hybridization
oven.
Approximately 20,000 transformants were screened
using this rapid method. Eighteen candidates were
identified which appeared to show increased ADH
thermotolerance. The candidates were purified and
assayed on the same filter as control strains (i.e.,
strain XL1 containing the LADH' plasmid pBPP, and strain
NM554 containing the LADH plasmid pBluescript).
Based on results of the filter screening, none of
the identified candidates appeared to have the
temperature-resistant phenotype suggested by the results
of the ADH filter assay. It is possible, however, that
thermoresistant mutants can be obtained with HA upon
further screening. Moreover, the chances of obtaining
mutagenized adh resulting in enzyme thermostabilization
might be further increased by excising the mutagenized
gene from the vector, and resubcloning into a wild-type
vector (i.e., a vector that has not been treated with
HA), followed by screening.
EXAMPLE 8: PCR Mutagenesis of the adh gene
This example describes PCR mutagenesis of the adh
gene as a representative alcohol dehydrogenase gene.
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To increase the efficiency of the cloning of
mutagenized adh, primers for directional cloning were
employed:
CCC CGA ATT CTC AAA ACG TCA GGA TGG TAC G ADH(EcoRI) [SEQ
5 ID N0:21]
CCC CTC TAG AAT AAA TGA GCA CAG CAG GAA AAG TAA TAA AAT
GC
ADH(XbaI) [SEQ ID N0:22]
The adh gene was amplified using these primers and cloned
10 into a pGEM-T vector.
For PCR mutagenesis two protocols were used, one
according to Spee et al. (Spee et al., Nucl. Acids Res.,
21, 777-778 (1993)), and another according to Rellos et
al., (Rellos et al., su ra) in which the limiting dNTP
15 concentration was double that of the first procedure and
dITP was not employed. The pGEM-T plasmid containing the
adh gene was then used as a template for PCR mutagenesis
of adh using standard T7 and SP6 primers to perform the
error-prone PCR reaction under these conditions.
20 Mutagenized adh-containing fragments were digested
using XbaI and EcoRI enzymes, and subcloned into
pBluescript SK to create a pBlue-ADH library. The
resultant pBlue-ADH library (i.e., one library for each
mutagenesis method performed) was transformed en masse
25 into E. coli strain NM554 to allow the adh gene to be
transcribed from the Iac promoter. Transformants were
then analyzed: (i) by PCR to determine the efficiency of
cloning (% of the plasmids with and without insert), and
ii) by ADH filter assay to determine the efficiency of
mutagenesis (% inactive ADH clones). The results of
these analyses are shown in Table 3.
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Table 3. Mutant candidates identified
Method of Percentage of the Percentage of the
mutagenesis* plasmids with the ADH' clones
insert
Method No. 1 60% 64%
Method No. 2 90% 36%
No mutagenesis 80% 75%
(wild-type adh)
* Method No.l was done according to Spee et al., su ra,
(i.e. with 14 ~.M of limiting dNTP and 200 ~M dITP) and
Method No. 2 was done according to Rellos et al., supra
(i.e. without dITP and with 25 ~.M of the limiting dNTP).
As can be seen from these results, both the cloning and
mutagenesis efficiency was better using the second
method.
The transformants were then plated to a density of
500 - 700 cells per plate and assayed on the filters
under the same conditions described in the prior example
for HA-mutagenesis of the adh gene. Approximately 5,000
clones containing adh mutagenized by the first method,
and the same number of clones mutagenized by the second
method, were tested. No thermostable candidates from the
first method were identified. By contrast, thirteen
candidates were selected from clones mutagenized by the
second method which appeared to possess an HLADH variant
that was more stable than the wild-type enzyme. Upon
restreaking and retesting these colonies by the filter
assay method, nine of the thirteen candidates (i.e.,
plasmids pAD7, pAD8, pADlO, pAD9l, pAD92, pAD93, pAD95,
pADlll, and pAD113) were chosen for further
characterization.
These results confirm that PCR-mediated mutagenesis,
particularly as described herein, can be employed to
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obtain potential thermostable LADH variants. The results
further indicate that the method can be employed to
obtain other stabilized alcohol dehydrogenases, or other
stabilized proteins.
S
EXAMPLE 9: Characterization of thermotolerant
HLADH candidates.
This example describes a characterization for
increased thermostability o~ mutants identified in the
prior example.
These experiments were done by calculating the
residual HLADH activity at 70°C for a series of
incubation periods. Residual activity is calculated as
activity after incubation at a particular temperature
divided by activity before incubation. Cultures of the
mutant candidates as well as control cells harboring the
wild-type HLADH+ control plasmid pBPP and HLADH- negative
control plasmid pGEM-T were grown in appropriate media,
and cell extracts were made by sonication. The extracts
were then incubated at 70°C, taking an initial sample
(to), and sampling at about 30, 60, and 120 minutes. The
samples were stored on ice, and the HLADH activity was
determined spectrophotometrically as described in Example
1. The data was plotted as a percentage of activity
compared to the to activity (residual activity) in order
to compare the individual samples to each other and
adjust for variations in expression levels or growth
variations.
Figure 4 displays the residual activity data for the
nine candidate plasmids pAD7, pADB, pADlO, pAD9l, pAD92,
pAD93, pAD95, pADlll, and pAD113, wherein the to activity
is normalized to 1.00 (100%). As can be seen from Figure
4, all the mutants exhibited increased thermotolerance
compared to cells containing plasmid pBPP, which contains
the wild-type HLADH gene. In particular, plasmids pAD9l,
pAD92, and pADlO showed the most noticeable alterations
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in thermostability. Cells containing pGEM-T (i.e., not
having an HLADH gene) did not show any HLADH activity.
These results thus confirm that the method of the
invention can be employed to obtain thermostable alcohol
dehydrase, particularly HLADH, mutants.
Table 4 below provides data illustrating comparative
data for HALDH activities in the original wild-type
("WT") clone and mutants. All clones were grown in 50 ml
of LB medium with 100 ~g/ml Amp (12.5 ug/ml Tet for WT
clone) overnight, concentrated in 1 ml of the assay
buffer (83 mM KHZPO4, 40 mM KC1, 0.25 mM EDTA), sonicated
and assayed with ethanol as a substrate and NAD cofactor,
with results shown as U = mol/mg protein x 1000 / percent
residual activity.
Table 4. HALDH Activity after Heat Treatment
Heat Treatment time
e~-r~;n R~r 15 min 30 min 60 min
pADH7 8 100% 4 50% 2 25% 0.6 8%
pADHB 21 100% 7.4 35% 2 10% 0.2 1%
pADHlO 16 100% 4 25% 1.4 9% 0 0%
pADH91 11 100% 8 73% 6 55% 4 36%
pADH92 25 100% 15 60% 17 68% 12 48%
pA.DH93 6 100% 1 17% 2.5 42% 0 0%
pADH95 66 100% 21 32% 10 15% 3 5%
pADHlll 22 100% 15 68% 16 73% 11 50%
pADH113 9 100% 4 44% 3 33% 0.8 9%
WT 10 100% 1 10% 0.3 3% 0 0%
Table 5 below provides data illustrating comparative
data for HALDH activities of the original wild-type
("WT") clone and mutants and substrate specificity. All
clones were grown in 1 L of LB medium with 100 ~g/ml Amp
(12.5 ~g/ml Tet for WT clone) overnight, concentrated in
50 ml of the assay buffer (83 mM KHzP04, 40 mM KC1, 0.25
mM EDTA), sonicated, incubated at 55°C for 5 min to
denature the E.coli protiens and lyophilized. The assays
were performed at room temperature with the listed
substrate and NAD cofactor, with results shown as U =
mol/mg protein x 1000.
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Table 5 HLADH Substrate Specificity
Strain Ethanol Isopropanol Butanol eenzyl Alcol
pADH7 8.7 0 5.3 1.4
pADHB 18.2 1.4 11 7
pADHlO 15.6 3 11.5 4.7
pADH91 13.2 1.1 4.7 3.4
pADH92 23.5 2.3 11 6.8
pADH93 5.6 1 3 1.6
pADH95 48 0.7 21.3 4.5
pADH111 22.6 1.6 9.8 3
pADH113 7 1.1 3 5
WT 9.2 1.7 7.6 3.5
Strain Hexanol Cvclohexanol R-(-)Butanol S-(+)Butanol
~ADH7 4 3 0 0
pADH8 _l5 49 2.4 2.2
pADHlO 15 69 10 4
pADH91 5.8 23 2.4 1.7
pADH92 10.6 50 2.3 2.4
pADH93 3.9 22 2 1.4
pADH95 21.3 16.5 0.5 0.8
pADHlll 9.4 58 4 2.7
pADH113 2.7 14.7 2 1.3
WT 10 42 4.3 2.9
EXAMPLE 10: Sequence Analysis of HLADH
Thermotolerant Candidates
This examples describes the sequencing of the
mutagenized adh genes.
The inserts of plasmids containing the mutagenized
adh gene were sequenced using an ABI DNA sequencer, and
compared to the sequence of the wild type protein. The
translated nucleic acid/amino acid sequence for plasmids
having the wild-type or mutant adh genes is given in
Figure 5, with the positions of the non-silent mutations
(i.e., those that change the encoded amino acid)
indicated by the boxes. Table 6 summarizes all the
nucleic acid mutations and the respective amino acid
changes, if any, introduced by the mutations.
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Table 6. Mutations identified in thermotolerant
candidates
Mutant Base Amino Original Mutant Amino
plasmid pair acid codon codon acid
position position' change2
pAD7 774 257 ATG ATA Met257I1e
878 292 GTG GCG Va1292A1a
pAD8 285 94 ACT ACC no as
change
806 268 GTC GCC Va1268A1a
pADlO 227 75 AGC AAC Ser75Asn
pAD91/92 284 94 ACT ATT Thr94Ile
pAD93 847 282 TGT AGT Cys282Ser
893 297 GAT GGT Asp297G1y
pAD95 774 257 ATG ATA Met257I1e
878 292 GTG GCG Va1292A1a
pADlll 532 177 TCT ACT Ser177Thr
pAD113 129 42 GCC GCT no as
change
159 52 GTG GTA no as
change
331 110 TTC CTC Phe110Leu
Also, the individual sequences of the mutant adh
5 sequences are set forth in the Sequence Listing for pAD7
(i.e., nucleic acid sequence at SEQ ID N0:3 and amino
acid sequence at SEQ ID N0:4), pAD8 (i.e., nucleic acid
sequence at SEQ ID N0:5 and amino acid sequence at SEQ ID
N0:6), pADlO (i.e., nucleic acid sequence at SEQ ID N0:7
10 and amino acid sequence at SEQ ID N0:8), pAD91/pAD92
(i.e., nucleic acid sequence at SEQ ID N0:9 and amino
acid sequence at SEQ ID NO:10), pAD93 (i.e., nucleic acid
sequence at SEQ ID NO:11 and amino acid sequence at SEQ
ID N0:12), pAD95 (i.e., nucleic acid sequence at SEQ ID
15 N0:13 and amino acid sequence at SEQ ID N0:14), pADlll
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(i.e., nucleic acid sequence at SEQ ID N0:15 and amino
acid sequence at SEQ ID N0:16), and pAD113(i.e., nucleic
acid sequence at SEQ ID N0:17 and amino acid sequence at
SEQ ID N0:18).
The first numbered amino acid in the wild-type and
mutant sequences is serine since, in the sequences
studied, the initial methionine (Met) is not present in
the final protein. However, it is possible that Met is
present in the wild-type (or mutant) HLADH sequences that
are produced in a different host, e.g., in a eukaryotic
host, or when transcribed and translated from a different
plasmid construct or chromosome.
As can be seen from this data, the sequences of
pAD91 and pAD92 are identical, which indicates the clones
from which the DNA was isolated likely are siblings.
Mutants containing plasmids pAD9l, PAD92, pAD93, and
pAD95 were identified from the same filter and mutants
containing plasmids pADll1 and pAD113 were identified
from the same filter assay. Also, in both pADB and
pAD91/92, the coding sequence specifying amino acid 94 is
mutated. Whereas this results in no change in this
position in pAD8, a mutation is introduced here in
pAD91/92. Similarly, two mutations in pAD113 are silent
and do not produce an amino acid change. These silent
mutations likely do not contribute substantially to the
thermostability of the protein.
EXAMPLE 11: Further thermostabilization
of HLADH proteins
This example describes the means by which the
thermostable proteins identified and characterized as in
the prior examples can be further thermostabilized.
Using the new mutants as a starting point, the
process applied here can be reiterated to increase the
thermostability of the HLADH enzyme even further.
Namely, it is expected that combinations of the
identified HLADH mutations or, combinations of these
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mutations with other HLADH mutations, can further
thermostabilize the enzyme.
In order to do this, the new thermoinactivation
limits need to be defined as described in Example 3.
This is followed by a new round of mutagenesis performed
as described in Examples 8, 9, and 10. In addition, the
identified mutations can be put together in differing
combinations by in vitro site-directed mutagenesis and
further molecular biology methods (see, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, NY. 1989)) that include DNA
shuffling via PCR methods (Stemmer et al., Proc. Natl.
Acad. Sci., 91, 10747-10751 (1994a); Stemmer et al.,
Nature, 340, 389-391 (1994b)}. As they have done in the
past, these methods are all expected to give further
increases in the levels of thermostability of the enzyme
or, in another similarly screened-for trait.
All of the references cited herein, including
patents, patent applications, sequences, and
publications, are hereby incorporated in their entireties
by reference.
While this invention has been described with an
emphasis upon preferred embodiments, it will be obvious
to those of ordinary skill in the art that variations in
the preferred embodiments can be used, including
variations due to improvements in the art, and that the
invention can be practiced otherwise than as specifically
described herein. Accordingly, this invention includes
all modifications encompassed within the spirit and scope
of the invention as defined by the following claims.
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SEQUENCE LISTING
(1)
GENERAL
INFORMATION:
S
(i) APPLICANT: DAVID C. DEMIRJIAN
IGOR A. BRIKUN
MALCOLM J. CASADAHAN
VERONIKA VONSTEIN
to
(ii) TITLE OF INVENTION: Method Proteins
For The Stabilization Of And
The
Thertnostabilized Alcohol ehydroge nases ProducedThereby
D
(iii) NUMBER OF SEQUENCES: 24
1S
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Mcdonald Berghoff
Boehnen Hulbert &
(B) STREET: 300 South blacker
Drive
(C) CITY: Chicago
20 (D) STATE: Illinois
(E) COUNTRY: United States
(F) ZIP: 60606
(v) COMPUTER READABLE FORM:
ZS (A) MEDIUM TYPE: Floppy
disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release
#1.0, Version #1.30
3 (vi) CURRENT APPLICATION DATA:
O
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
3S
(2)
INFORMATION
FOR
SEQ
ID
NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
!~~ (B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
4S
(xi) SEQUENCE DESCRIPTION: SEQ
ID NO:1:
ATG ACA GCA GGA AAA GTA ATA GCTGTGCTGTGG 48
AGC AAA TGC AAA GCG
S Ser Thr Ala Gly Lys Val Ile AlaValLeuTrp
~ Lys Cys Lys Ala
1 5 10 15
GAG AAG AAA CCA TTT TCC ATC GTTGCACCCCCG 96
GAA GAG GAG GTG GAG
Glu Lys Lys Pro Phe Ser Ile ValAlaProPro
Glu Glu Glu Val Glu
SS 2p 25 30
AAG CAT GAA GTC CGT ATA AAG GGAATTTGTCGC 144
GCC ATG GTG GCC ACA
Lys His Glu Val Arg Ile Lys GlyIleCysArg
Ala Met Val Ala Thr
35 40 45
6O
TCA GAC CAC GTG GTT AGT GGA CCTCTTCCTGTG 192
GAT ACC CTT GTC ACA
Ser Asp His Val Val Ser Gly ProLeuProVal
Asp Thr Leu Val Thr
50 55 60
C)S ATC GGC CAT GAG GCA GCG GGC ATTGGAGAAGGC 240
GCA ATT GTG GAG AGC
Ile Gly His Glu Ala Ala Gly IleGlyGluGly
Ala Ile Val Glu Ser
65 '10 '15
GTC ACA GTA AGA CCA GGT GAT CTCTTTACTCCC 288
ACT AAA GTC ATC CCA
Val Thr Val Arg Pro Gly Asp LeuPheThrPro
Thr Lys Val Ile Pro
gp 85 90 95
CAG GGA AAA TGC AGG GTT TGT GGCAACTTCTGC 336
TGT AAG CAC CCT GAA
Gln Gly Lys Cys Arg Val Cys GlyAsnPheCys
Cys Lys His Pro Glu
7S 100 105 110
TTG AAT GAT CTG AGC ATG CCT CAGGATGGTACC 384
AAA CGG GGA ACC ATG
Leu Asn Asp Leu Ser Met Pro GlnAspGlyThr
Lys Arg Gly Thr Met
115 120 125
8
0
AGC TTC ACC TGC AGA GGG AAG TTCCTTGGC 432
AGG CCC ATC CAC CAC ACC
Ser Phe Thr Cys Arg Gly Lys PheLeuGly
Arg Pro Ile His His Thr
130 135 140
SUBSTITUTE SHEET (RULE 26)
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34
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
195 I50 155
S ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
GCC AAG GTC ACC CAG
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
GTG GGC CTG TCT GTT
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly
Val Gly Leu Ser Val
1S 19s zoo 2os
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala G1y Ala Ala Arg
Ile Ile Gly Val Asp
210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
225 230 235
2S TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
GAA GTC ATT GGT CGG
3 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe
0 Glu Val Ile Gly Arg
260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT 864
CAA GAA GCA TAT GGT
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys
Gln Glu Ala Tyr Gly
3S 27s 2so zes
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val Ile Val Gly Val Pro Pro Asp Ser
Gln Asn Leu Ser Met
290 295 300
40
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG 960
AAA GGA GCT ATT TTT
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
Lys Gly Ala Ile Phe
305 3I0 315
4S GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA 1008
CTT GTG GCC GAT TTT
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys
Leu Val Ala Asp Phe
320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC 1056
ACC CAT GTT TTA CCT
S Met Ala Lys Lys Phe Ala Leu Asp Pro Leu I1e
0 Thr His Val Leu Pro
340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT 1104
CGC TCT GGA GAG AGT
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu
Arg Ser Gly Glu Ser
SS 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Ile Leu Thr Phe
370
60
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
6S (A) LENGTH: 374 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
,70
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp Glu
,7S1 5 10 15
Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu
Val Ala Pro Pro Lys
20 25 30
Ala His Glu Val Arg Ile Lys Met Val Ala Thr
Gly Ile Cys Arg Ser
g0 35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr
Pro Leu Pro Val Ile
50 55 60
SUBSTITUTE SHEET (RULE 26)
~ T
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val
65 70 75 80
Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln
.S 85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu
100 105 110
10 Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
I Jr 130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys ~eu Ile Gly Cys Gly Phe
20 lss 170 17s
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
180 185 190
25 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg !le Ile Gly Val Asp Ile
30 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys
225 230 235 240
Val Asn Pro Gln Asp Tyr Lys Lys pro Ile Gln Glu Val Leu Thr Glu
35 245 250 255
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu
260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val
275 280 285
Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn
290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly
305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met
325 330 335
Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe
340 345 350
SS Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile
355 360 365
Arg Thr Ile Leu Thr Phe
370
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
7S ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96
g 0 Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro
20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144
Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg
SUBSTITUTE SHEET (RULE 26)
i i i
CA 02290074 1999-11-12
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36
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC 192
ACA CCT CTT CCT GTG
Ser Asp Asp His Val Val Ser Gly Thr Leu Val
Thr Pro Leu Pro Val
S 50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
Ser Ile Gly Glu Gly
65 70 75
IO GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
80 85 90 95
IS CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
ATG CAG GAT GGT ACC
ZO Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
Met Gln Asp Gly Thr
115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
His Phe Leu Gly Thr
?.S130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
145 150 155
3 ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
O CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
3 TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
S GCC AAG GTC ACC CAG
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
GTG GGC CTG TCT GTT
4O Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly
Val Gly Leu Ser Val
195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg
Ile Ile Gly Val Asp
G~S210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
225 230 235
S TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
O CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
S GAA ATA AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
S GAA GTC ATT GGT CGG
Glu Ile Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Val Ile Gly Arg
260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT 864
CAA GAA GCA TAT GGT
6O Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys
Gln Glu Ala Tyr Gly
275 280 285
GTG AGC GTC ATT GCG GGA GTA CCT CCT GAT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val Ile Ala Gly Val Pro Pro Asp Ser
Gln Asn Leu Ser Met
65 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG 960
AAA GGA GCT ATT TTT
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
Lys Gly Ala Ile Phe
305 310 315
7 GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA 1008
O CTT GTG GCC GAT TTT
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys
Leu Val Ala Asp Phe
320 325 330 335
7S ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC 1056
ACC CAT GTT TTA CCT
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile
Thr His Val Leu Pro
340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT 1104
CGC TCT GGA GAG AGT
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu
Arg Ser Gly Glu Ser
355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile P.rg Thr Ile Leu Thr Phe
SUBSTITUTE SHEET (RULE 26)
'I I 1
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
37
370
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acid:.
(B) TYPE: amino acid
to (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
IS Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu
1 s to is
Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys
20 20 25 30
Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser
35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile
25 SO 55 60
Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val
65 70 75 80
3 0 Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu
35 loo los 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
40 130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
45 Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
50 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile
55 210 215 220
Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys
225 230 235 240
60Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu
245 250 255
Ile SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu
65 260 265 270
Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal
275 280 285
Ser ValIleAlaGlyValProProAspSerGlnAsnLeuSerMetAsn
70 290 295 300
Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly
305 310 315 320
75Gly PheLysSerLysAapSerValProLysLeuValAlaAspPheMet
325 330 335
Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe
340 345 350
Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle
355 360 365
Arg ThrIleLeuThrPhe
SUBSTITUTE SHEET (RULE 26)
i i i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
38
370
(2) INFORMATION FOR SEQ ID NO: S:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA 48
GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp
1 5 10 15
ZO
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG 96
GAG GTT GCA CCC CCG
Glu Glu Lys Lys Pro Phe Ser Ile G1u Glu Val
Glu Val Ala Pro Pro
20 25 30
?.S AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144
ACA GGA ATT TGT CGC
Lys Ala His Glu Val Arg Ile Lys Met Val Ala
Thr Gly Ile Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC 192
ACA CCT CTT CCT GTG
3 Ser Asp Asp His Val Val Ser Gly Thr Leu Val
~ Thr Pro Leu Pro Val
SO SS 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
Ser Ile Gly Glu Gly
3S 6s 70 7s
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACC CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
g0 85 90 95
4O
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
4S TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
ATG CAG GAT GGT ACC
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
Met Gln Asp Gly Thr
115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Sfl Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
His Phe Leu Gly Thr
130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
SS 14s lso lss
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
6O
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
GCC AAG GTC ACC CAG
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
C)S GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
GTG GGC CTG TCT GTT
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly
Val Gly Leu Ser Val
195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala Gly Ala A1a Arg
Ile Ile Gly Val Asp
210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
7S 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
go
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
GAA GCC ATT GGT CGG
Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Ala Ile Gly Arg
260 265 270
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
39
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT CAA GAA GCA TAT GGT 864
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln G1u A1a Tyr Gly
275 280 285
S GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC CAA AAT CTC TCT ATG 912
Val Ser Val ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met
290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe
305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe
IS 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro
340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser
355 360 365
2S ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Ile Leu Thr Phe
370
3 O (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids
(B) TYPE: amino acid
3 5 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu
1 5 10 15
Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys
4S zo zs 30
Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser
35 40 45
S O Asp Asp His Val val Ser Gly Thr Leu Val Thr Pro Leu Pro val Ile
55 60
Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val
65 70 75 80
SS
Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu
loo los llo
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
6S Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
7S 180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
g O Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile
210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys
225 230 235 240
SUBSTITUTE SHEET (RULE 26)
i i i
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WO 98/51802 PCT/US98/09627
Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln
Glu Val Leu Thr Glu
245 250 255
S Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu
Ala Ile Gly Arg Leu
260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln
Glu Ala Tyr Gly Val
275 280 285
10
Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln
Asn Leu Ser Met Asn
290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys
Gly Ala Ile Phe Gly
IS 305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu
Val Ala Asp Phe Met
325 330 335
20 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr
His Val Leu Pro Phe
340 345 350
Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg
Ser Gly Glu Ser Ile
355 360 365
25
Arg Thr Ile Leu Thr Phe
370
3 (2) INFORMATION FOR SEQ ID N0:7:
O
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(8) TYPE: nucleic acid
35 (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
40
(xi1 SEQUENCE DESCRIPTION: SEQ ID N0:7:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA 48
GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp
4S 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG 96
GAG GTT GCA CCC CCG
Glu Glu Lys Lys Fro Phe Ser Ile Glu Glu Val
Glu Val Ala Pro Pro
20 25 30
S
O
~G GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144
ACA GGA ATT TGT CGC
Lys Ala His Glu Val Arg Ile Lys Met Val Ala
Thr Gly Ile Cys Arg
35 40 45
S TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC 192
S ACA CCT CTT CCT GTG
Ser Asp Asp His Val Val Ser Gly Thr Leu Val
Thr Pro Leu Pro Val
SS 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AAC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
Asn Ile Gly Glu Gly
70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
65 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
7O
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
ATG CAG GAT GGT ACC
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
Met Gln Asp Gly Thr
115 120 125
7S AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
His Phe Leu Gly Thr
130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
80 Ser Thr Phe Ser Gln Tyr Thr Val Va1 Asp Glu
Ile Ser Val Ala Lys
145 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
SUBSTITUTE SHEET (RULE 25)
( i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
41
160 165 170 175
TTTTCTACTGGTTATGGGTCTGCAGTCAAGGTTGCC GTCACCCAG 576
AAG
PheSerThrGly GlySerAlaValLysValAlaLysValThrGln
i8o 185 190
GGCTCCACCTGTGCCGTGTTTGGCCTTGGAGGAGTGGGCCTGTCTGTT 624
GlySerThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerVal
195 200 205
1~
ATCATGGGCTGTAAAGCAGCCGGAGCGGCCAGGATCATTGGGGTGGAC 672
IleMetGlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAsp
210 215 22p
IS ATCAACAAAGACAAGTTTGCAAAGGCCAAAGAAGTGGGTGCCACTGAG 720
IleAsnLysAspLysPheAlaLysAlaLysGluValGlyA1aThrGlu
225 230 235
TGTGTCAACCCTCAGGACTACAAGAAACCCATCCAGGAGGTGCTGACA 768
20 CysValAsnProGlnAspTyrLysLysProIleGlnGluValLeuThr
240 245 250 255
GAAATGAGCAATGGAGGTGTGGATTTTTCCTTTGAAGTCATTGGTCGG 816
GluMetSerAsnGlyGlyValAspPheSerPheGluValIleGlyArg
25 zso 265 270
CTCGACACTATGGTGACTGCCTTGTCATGCTGTCAAGAAGCATATGGT 864
LeuAspThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGly
30 275 280 285
GTGAGCGTCATTGTGGGAGTACCTCCTGATTCCCAAAATCTCTCTATG 912
ValSerValIleValGlyValProProAspSerGlnAsnLeuSerMet
290 295 300
3S AATCCTATGTTGCTACTGAGTGGACGTACCTGGAAAGGAGCTATTTTT 960
AsnProMetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePhe
305 310 315
GGCGGTTTTAAGAGTAAAGATTCTGTCCCCAAACTTGTGGCCGATTTT 1008
40 GlyGlyPheLysSerLysAspSerValProLysLeuValAlaAspPhe
320 325 330 335
ATGGCTAAAAAGTTTGCACTGGATCCTTTAATCACCCATGTTTTACCT 1056
MetAlaLysLysPheAlaLeuAspProLeuIleThrHisValLeuPro
45 340 345 350
TTTGAAAAAATAAATGAAGGATTTGACCTGCTTCGCTCTGGAGAGAGT 1104
PheGluLysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSer
50 355 360 365
ATCCGTACCATCCTGACGTTTTGA 1128
IleArgThrIleLeuThrPhe
370
SS
(2)INFORMAT IONFORSEQID
N0:8:
( i) EQUENCE CHARACTE RISTICS:
S
(A)LENGTH: 374aminocids
a
(B)TYPE:minoacid
a
(D)TOPOLOGY: inear
l
(i i) TYPE: otein
MOLECULE pr
6S (x i) EQUENCE DESCRIPT ION:SEQ N0:8:
S ID
SerThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu
1 S 10 15
70 GluLysLysProPheSerIleGluGluValGluValAlaProProLys
20 25 30
AlaHisGluValArgIleLysMetValAlaThrGlyIleCysArgSer
75 35 40 q5
AspAspHisValValSerGlyThrLeuValThrProLeuProValIle
50 55 60
AlaGlyHisGluAlaAlaGlyIleValGluAsnIleGlyGluGlyVal
65 70 75 80
ThrThrValArgProGlyAspLysValIleProLeuPheThrProGln
85 90 95
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
42
Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu
100 105 110
Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer
115 120 125
Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer
130 135 140
Thr PheSerGlnTyrThrValValAspGluIleSerValAlaLysIle
145 150 155 160
Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe
165 170 175
Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrGlnGly
180 185 190
Ser ThrCysAIaValPheGlyLeuGlyGlyValGlyLeuSerValIle
195 200 205
Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle
210 215 220
Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys
225 230 235 240
Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu
245 250 255
Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu
260 265 270
Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal
275 2eo 28s
Ser ValIleValGlyValProProAspSerGlnAsnLeuSerMetAsn
290 295 300
Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly
305 310 315 320
Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet
325 330 335
Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe
340 345 350
Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle
355 360 365
Arg ThrIleLeuThrPhe
370
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96
Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro
20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144
Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192
Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val
50 55 60
SUBSTITUTE SHEET (RULE 2fi)
'f I T
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
43
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
Ser Ile Gly Glu Gly
65 70 75
S GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ATT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Ile Pro
BO 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
ATG CAG GAT GGT ACC
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
1 Met Gln Asp Gly Thr
S 115
120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
His Phe Leu Gly Thr
130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
145 150 155
ZS ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
3 GCC AAG GTC ACC CAG
0
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
GTG GGC CTG TCT GTT
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly
3S Val Gly Leu Ser Val
195
200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg
Ile Ile Gly Val Asp
40 21o zls z2o
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
225 230 235
4S TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
S GAA GTC ATT GGT CGG
0
Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Val Ile Gly Arg
260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT 864
CAA GAA GCA TAT GGT
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys
Gln Glu Ala Tyr Gly
SS 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val Ile Val Gly Val Pro Pro Asp Ser
Gln Asn Leu Ser Met
60 290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG 960
AAA GGA GCT ATT TTT
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
Lys Gly Ala Ile Phe
305 310 315
6S GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA 1008
CTT GTG GCC GAT TTT
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys
Leu Val Ala Asp Phe
320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC 1056
70 ACC CAT GTT TTA CCT
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile
Thr His Val Leu Pro
340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT 1104
CGC TCT GGA GAG AGT
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu
Arg Ser Gly Glu Ser
7S 355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Zle Leu Thr Phe
370
80
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
44
(A) acids
LENGTH:
374
amino
(B ) amino id
TYPE: ac
(D ) linear
TOPOLOGY:
S ( ii)MOLECULETYPE: rotein
p
( xi)SEQUENCEDESCRIPTION : Q NO:10:
SEID
Ser ThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu
I 1 5 10 15
O
Glu LysLysProPheSerIleGluGluValGluValAlaProProLys
20 25 30
IS Ala HisGluValArgIleLysMetValAlaThrGlyIleCysArgSer
35 40 45
Asp AspHisValValSerGlyThrLeuValThrProLeuProValIle
20 so ss 60
Ala GlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal
65 70 7s 80
Thr ThrValArgProGlyAspLysValIleProLeuPheIleProGln
25 es 90 9s
Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu
100 105 110
3 Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer
O
115 120 125
Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer
35 130 135 140
Thr PheSerGlnTyrThrValValAspGlu_TleSerValAlaLysIle
145 150 155 160
Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe
40 16s 170 17s
Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrGlnGly
180 185 190
45 Ser ThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerValIle
195 200 20s
Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle
50 0 215 220
Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys
225 230 235 240
Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu
55 245 250 25s
Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu
260 265 270
60 Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal
275 28Q 285
Ser ValIleValGlyValProProAspSerGlnAsnLeuSerMetAsn
65 29 295 300
Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly
305 310 315 320
Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet
70 325 330 335
Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe
340 345 350
75 Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle
355 360 365
Arg ThrIleLeuThrPhe
8O 370
(2) INFORMATION FOR SEQ ZD NO:11:
(i) SEQUENCE CHARACTERISTICS:
SUBSTITUTE SHEET (RULE 26)
r ~ t
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
4S
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
S (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
IO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA 48
GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp
1 5 10 15
IS GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG 96
GAG GTT GCA CCC CCG
Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val
Glu Val Ala Pro Pro
20 25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144
20 ACA GGA ATT TGT CGC
l
i
Lys A
a H
s Glu Val Arg Ile Lys Met Val Ala Thr Gly
Ile Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC 192
ACA CCT CTT CCT GTG
Ser Asp Asp His Val Val Ser Gly Thr Leu Val
2S Thr Pro Leu Pro Val
o
s
ss so
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Giu
Ser Ile Gly Glu Gly
30 65 7 7s
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
80 85 90 95
3 CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
S GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
40 ATG CAG GAT GGT ACC
L
L
eu
ys Asn Asp Leu Ser Met Pro Arg Gly Thr Met
Gln Asp Gly Thr
115 120 125
AGC AGG TTC ACC T'GC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
4S His Phe Leu Gly Thr
13 0
13 5 14 0
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
SO 1 5 150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
SS TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
GCC AAG GTC ACC CAG
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
60 GTG GGC CTG TCT GTT
Gl
S
h
y
er T
r Cys Ala Val Phe Gly Leu Gly Gly Val Gly
Leu Ser Val
195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg
6S Ile Ile Gly Val Asp
2
215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
7O 225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
IS GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
GAA GTC ATT GGT CGG
Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Val Ile Gly Arg
260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC AGT 864
g0 CAA GAA GCA TAT GGT
A
Th
sp
Leu
r Met Val Thr Ala Leu Ser Cys Ser Gln Glu
Ala Tyr Gly
275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GGT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val Ile Val Gly Val Pro Pro Gly Ser
G1n Asn Leu Ser Met
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
46
290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA GGA GCT ATT TTT 960
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe
305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu val Ala Asp Phe
l0 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro
340 395 350
IS TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser
355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Ile Leu Thr Phe
370
(2) INFORMATION FORSEQID
N0:12:
25
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH :
374
amino
acids
(B)TYPE: amino
acid
(D)TOPOLOGY:
linear
3
O
(ii) TYPE:
MOLECULE protein
(xi) DESCRIPTION: SEQ N0:12:
SEQUENCE ID
35 Ser ThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu
1 5 10 15
Glu LysLysProPheSerIleGluGluValGluValAlaProProLys
20 25 30
40
Ala HisGluValArgIleLysMetValAlaThrGlyIleCysArgSer
35 40 45
Asp AspHisValValSerGlyThrLeuValThrProLeuProValIle
45 50 55 60
Ala GlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal
65 70 75 80
Thr ThrValArgProGlyAspLysValIleProLeuPheThrProGln
85 90 95
Cys GlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu
100 105 110
55
Lys AsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThrSer
115 120 125
Arg PheThrCysArgGlyLysProIleHisHisPheLeuGlyThrSer
60 130 135 140
Thr PheSerGlnTyrThrValValAspGluIleSerValAlaLysIle
145 150 155 160
65 Asp AlaAlaSerProLeuGluLysValCysLeuIleGlyCysGlyPhe
165 170 I75
Ser ThrGlyTyrGlySerAlaValLysValAlaLysValThrClnGly
180 185 190
Ser ThrCysAlaValPheG1yLeuGlyGlyValGlyLeuSerValIle
195 200 205
Met GlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAspIle
75 210 215 220
Asn LysAspLysPheAlaLysAlaLysGluValGlyAlaThrGluCys
225 230 235 240
80 Val AsnProGlnAspTyrLysLysProIleGlnGluValLeuThrGlu
245 250 255
Met SerAsnGlyGlyValAspPheSerPheGluValIleGlyArgLeu
260 265 270
SUBSTITUTE SHEET (RULE 26)
r m
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
47
Asp ThrMetValThrAlaLeuSerCysSerGlnGluAlaTyrGlyVal
275 280 285
S Ser ValIleValGlyValProProGlySerGlnAsnLeuSerMetAsn
290 295 300
Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly
305 310 315 320
Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet
325 330 335
Ala LysLysPheAlaLeuAspProLeuileThrHisValLeuProPhe
1
S
340 345 350
Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle
355 360 365
Z0 Arg ThrIleLeuThrPhe
370
2 S (2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
3 0 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
3 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
S
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA 48
GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp
40 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG 96
GAG GTT GCA CCC CCG
Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val
Glu Val Ala Pro Pro
25 30
4S AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC 144
ACA GGA ATT TGT CGC
Lys Ala His Glu Val Arg Ile Lys Met Val Ala
Thr Gly Ile Cys Arg
35 40 45
TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC 192
S ACA CCT CTT CCT GTG
0
Ser Asp Asp His Val Val Ser Gly Thr Leu Val
Thr Pro Leu Pro Val
50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
SS Ser Ile Gly Glu Gly
6s
70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
60 80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC TTC TGC
Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
Glu Gly Asn Phe Cys
100 105 110
C)STTG AAA AAT GAT CTG AGC ATG CCT CGG rGA ACC 389
ATG CAG GAT GGT ACC
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
Met Gln Asp Gly Thr
115 120 125
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
70 CAC TTC CTT GGC ACC
h
h
Ser Arg P
e T
r Cys Arg Gly Lys Pro Ile His His Phe Leu Gly
Thr
130 135 140
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
7S Ile Ser Val Ala Lys
145
150 155
ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
g0 160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
GCC AAG GTC ACC CAG
Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
Ala Lys Val Thr Gln
180 185 190
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
48
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA GTG 624
GGC CTC TCT GTT
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val
Gly Leu Ser Val
195 200 205
S ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
I1e Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile
Ile Gly Val Asp
210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA GTG 720
GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
225 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC CAG 768
GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln
Glu Val Leu Thr
~S 240 245 250 255
GAA ATA AGC AAT GGA GGT GTG GAT TTT TCC TTT GAA 816
GTC ATT GGT CGG
Glu GCG Ser Asn Gly Gly Val Asp Phe Ser Phe Glu
Val Ile Gly Arg
260 265 270
ZO CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT 864
CAA GAA GCA TAT GGT
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln
Glu Ala Tyr Gly
275 280 285
ZS GTG AGC GTC ATT GCG GGA GTA CCT CCT GAT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val ile Ala Gly Val Pro Pro Asp Ser Gln
Asn Leu Ser Met
290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG AAA 960
GGA GCT ATT TTT
3 O Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
Lys Gly Ala Ile Phe
305 310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT 1008
GTG GCC GAT TTT
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu
Val Ala Asp Phe
3 S 320 325 330 335
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC 1056
CAT GTT TTA CCT
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr
His Val Leu Pro
390 345 350
4O TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT 1104
CGC TCT GGA GAG AGT
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg
Ser Gly Glu Ser
355 360 365
4S ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Ile Leu Thr Phe
370
SO (2) INFORMATION FOR SEQ ID N0:14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids
(B) TYPE: amino acid
SS (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi1 SEQUENCE DESCRIPTION: SEQ ID N0:14:
6O Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu
1 5 10 15
Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys
6S 20 25 30
Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser
35 40 45
70 Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile
50 55 60
Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val
65 70 75 80
7S Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln
85 90 95
gO Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Phe Cys Leu
loo los zlo
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
SUBSTITUTE SHEET (RULE 26)
I I T
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
49
Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
S 145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
165 170 175
IO Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
IS Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Iie
210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys
ZO 225 230 235 240
Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu
245 250 255
ZS Ile Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu
260 265 270
Asp ThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGlyVal
275 280 285
3
O
Ser ValIleAlaGlyValProProAspSerGlnAsnLeuSerMetAsn
290 295 300
Pro MetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePheGly
3S 305 310 315 320
Gly PheLysSerLysAspSerValProLysLeuValAlaAspPheMet
325 330 335
4O Ala LysLysPheAlaLeuAspProLeuIleThrHisValLeuProPhe
340 345 350
Glu LysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSerIle
355 360 365
45
Arg ThrIleLeuThrPhe
370
SO (2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
SS (C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
6O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA GCG GCT GTG CTG TGG 48
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp
6S 1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG GAG GTT GCA CCC CCG 96
Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro
20 25 30
7O AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCC ACA GGA ATT TGT CGC 144
Lys Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg
35 40 45
7S TCA GAT GAC CAC GTG GTT AGT GGA ACC CTT GTC ACA CCT CTT CCT GTG 192
Ser Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val
50 55 60
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG AGC ATT GGA GAA GGC 240
8O Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly
65 70 75
GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC CCA CTC TTT ACT CCC 288
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro
SUBSTITUTE SHEET (RULE 26)
i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
SO
80 BS 90 95
CAG TGTGGAAAATGCAGGGTTTGTAAGCACCCTGAAGGCAACTTCTGC 336
Gln CysGlyLysCysArgValCysLysHisProGluGlyAsnPheCys
S 100 105 110
TTG AAAAATGATCTGAGCATGCCTCGGGGAA~CATGCAGGATGGTACC 384
Leu LysAsnAspLeuSerMetProArgGlyThrMetGlnAspGlyThr
lls 120 lzs
AGC AGGTTCACCTGCAGAGGGAAGCCCATCCACCACTTCCTTGGCACC 432
Ser ArgPheThrCysArgGlyLysProIleHisHisPheLeuGlyThr
130 135 140
IS AGC ACCTTCTCCCAGTACACCGTGGTGGACGAGATCTCAGTGGCCAAG 480
Ser ThrPheSerGlnTyrThrValValAspGluIleSerValAlaLys
145 150 155
ATC GATGCGGCCTCACCGCTGGAGAAAGTCTGTCTCATTGGCTGTGGA 528
Ile AspAlaAlaSerProLeuGluLysValCysLeuIleGlyCysGly
160 165 170 175
TTT ACTACTGGTTATGGGTCTGCAGTCAAGGTTGCCAAGGTCACCCAG 576
Phe ThrThrGlyTyrGlySerAlaValLysValAlaLysValThrGln
ZS 180 185 190
GGC TCCACCTGTGCCGTGTTTGGCCTTGGAGGAGTGGGCCTGTCTGTT 624
Gly SerThrCysAlaValPheGlyLeuGlyGlyValGlyLeuSerVal
195 200 205
ATC ATGGGCTGTAAAGCAGCCGGAGCGGCCAGGATCATTGGGGTGGAC 672
Ile MetGlyCysLysAlaAlaGlyAlaAlaArgIleIleGlyValAsp
210 215 220
3 ATC AACAAAGACAAGTTTGCAAAGGCCAAAGAAGTGGGTGCCACTGAG 720
S
Ile AsnLysAspLysPheAlaLysAlaLysGluValGlyAlaThrGlu
225 230 235
TGT GTCAACCCTCAGGACTACAAGAAACCCATCCAGGAGGTGCTGACA 768
Cys ValAsnProGlnAspTyrLysLysProIleGlnGluValLeuThr
240 245 250 255
GAA ATGAGCAATGGAGGTGTGGATTTTTCCTTTGAAGTCATTGGTCGG 816
Glu MetSerAsnGlyGlyValAspPheSerPheGluValIleGlyArg
4S 260 265 270
CTC GACACTATGGTGACTGCCTTGTCATGCTGTCAAGAAGCATATGGT 864
Leu AspThrMetValThrAlaLeuSerCysCysGlnGluAlaTyrGly
275 280 285
SO
GTG AGCGTCATTGTGGGAGTACCTCCTGATTCCCAAAATCTCTCTATG 912
Val SerValIleValGlyValProProAspSerGlnAsnLeuSerMet
290 295 300
SS AAT CCTATGTTGCTACTGAGTGGACGTACCTGGAAAGGAGCTATTTTT 960
Asn ProMetLeuLeuLeuSerGlyArgThrTrpLysGlyAlaIlePhe
305 310 315
GGC GGTTTTAAGAGTAAAGATTCTGTCCCCAAACTTGTGGCCGATTTT 1008
60 Gly GlyPheLysSerLysAspSerValProLysLeuValAlaAspPhe
320 325 330 335
ATG GCTAAAAAGTTTGCACTGGATCCTTTAATCACCCATGTTTTACCT 1056
Met AlaLysLysPheAlaLeuAspProLeuIleThrHisValLeuPro
6S 340 345 350
TTT GAAAAAATAAATGAAGGATTTGACCTGCTTCGCTCTGGAGAGAGT 1104
Phe GluLysIleAsnGluGlyPheAspLeuLeuArgSerGlyGluSer
355 360 365
,70
ATC CGTACCATCCTGACGTTTTGA 1128
Ile ArgThrIleLeuThrPhe
370
7S
(2) INFORMATION FORSEQID
N0:16:
(i)SEQUENCE CHARACTERISTICS :
(A)LENGTH : 4
37amino
acids
g (B)TYPE:
0 amino
acid
(D)TOPOLOGY: linear
(ii) MOLECULE TYPE: rotein
p
SUBSTITUTE SHEET (RULE 26)
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
51
(xi) DESCRIPTION: SEQIDN0:16:
SEQUENCE
SerThrAlaGlyLysValIleLysCysLysAlaAlaValLeuTrpGlu
1 5 10 15
GluLysLysProPheSerIleGluGluValGluValAlaProProLys
20 25 30
AlaHisGluValArgIleLysMetValAlaThrGlyIleCysArgSer
35 40 45
AspAspHisValValSerGlyThrLeuValThrProLeuProValIle
50 55 60
IS AlaGlyHisGluAlaAlaGlyIleValGluSerIleGlyGluGlyVal
65 70 75 80
ThrThrValArgProGlyAspLysValIleProLeuPheThrProGln
85 90 95
2o
CysGlyLysCysArgValCysLysHisProGluGlyAsnPheCysLeu
100 105 110
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
75 115 120 125
Azg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
130 135 140
3 d Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
165 170 175
35 Thr Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Va1 Thz Gln Gly
180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
40 195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile
210 215 220
4 5 Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val GIy Ala Thr Glu Cys
225 230 235 240
Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu
245 250 255
5o Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu
260 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val
55 z75 2so zss
Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn
290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly
305 310 315 320
Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met
325 330 335
65 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe
340 345 350
Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile
355 360 365
Arg Thr Ile Leu Thr Phe
370
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1128 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
SUBSTITUTE SHEET (RULE 26)
i i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
S2
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
S ATG AGC ACA GCA GGA AAA GTA ATA AAA TGC AAA 48
GCG GCT GTG CTG TGG
Ser Thr Ala Gly Lys Val Ile Lys Cys Lys Ala
Ala Val Leu Trp
1 5 10 15
GAG GAA AAG AAA CCA TTT TCC ATC GAG GAG GTG 96
GAG GTT GCA CCC CCG
Glu Glu Lys Lys Pro Phe Ser Ile Glu Glu Val
Glu Val Ala Pro Pro
25 30
AAG GCC CAT GAA GTC CGT ATA AAG ATG GTG GCT 144
ACA GGA ATT TGT CGC
Lys Ala His Glu Val Arg Ile Lys Met Val Ala
Thr Gly Ile Cys Arg
1S 35 40 45
TCA GAT GAC CAC GTA GTT AGT GGA ACC CTT GTC 192
ACA CCT CTT CCT GTG
Ser Asp Asp His Val Val Ser Gly Thr Leu Val
Thr Pro Leu Pro Val
50 55 60
20
ATC GCA GGC CAT GAG GCA GCG GGC ATT GTG GAG 240
AGC ATT GGA GAA GGC
Ile Ala Gly His Glu Ala Ala Gly Ile Val Glu
Ser Ile Gly Glu Gly
65 70 75
2S GTC ACT ACA GTA AGA CCA GGT GAT AAA GTC ATC 288
CCA CTC TTT ACT CCC
Val Thr Thr Val Arg Pro Gly Asp Lys Val Ile
Pro Leu Phe Thr Pro
80 85 90 95
CAG TGT GGA AAA TGC AGG GTT TGT AAG CAC CCT 336
GAA GGC AAC CTC TGC
3 Gln Cys Gly Lys Cys Arg Val Cys Lys His Pro
0 Glu Gly Asn Leu Cys
100 105 110
TTG AAA AAT GAT CTG AGC ATG CCT CGG GGA ACC 384
ATG CAG GAT GGT ACC
Leu Lys Asn Asp Leu Ser Met Pro Arg Gly Thr
Met Gln Asp Gly Thr
3S lls lzo 12s
AGC AGG TTC ACC TGC AGA GGG AAG CCC ATC CAC 432
CAC TTC CTT GGC ACC
Ser Arg Phe Thr Cys Arg Gly Lys Pro Ile His
His Phe Leu G1y Thr
130 135 140
40
AGC ACC TTC TCC CAG TAC ACC GTG GTG GAC GAG 480
ATC TCA GTG GCC AAG
Ser Thr Phe Ser Gln Tyr Thr Val Val Asp Glu
Ile Ser Val Ala Lys
145 150 155
4S ATC GAT GCG GCC TCA CCG CTG GAG AAA GTC TGT 528
CTC ATT GGC TGT GGA
Ile Asp Ala Ala Ser Pro Leu Glu Lys Val Cys
Leu Ile Gly Cys Gly
160 165 170 175
TTT TCT ACT GGT TAT GGG TCT GCA GTC AAG GTT 576
GCC AAG GTC ACC CAG
S Phe Ser Thr Gly Tyr Gly Ser Ala Val Lys Val
0 Ala Lys Val Thr Gln
180 185 190
GGC TCC ACC TGT GCC GTG TTT GGC CTT GGA GGA 624
GTG GGC CTG TCT GTT
Gly Ser Thr Cys Ala Val Phe Gly Leu Gly Gly
Val Gly Leu Ser Val
SS 195 200 205
ATC ATG GGC TGT AAA GCA GCC GGA GCG GCC AGG 672
ATC ATT GGG GTG GAC
Ile Met Gly Cys Lys Ala Ala Gly Ala Ala Arg
Ile Ile Gly Val Asp
210 215 220
60
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
Val Gly Ala Thr Glu
225 230 235
6S TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
240 245 250 255
GAA ATG AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
GAA GTC ATT GGT CGG
70 Glu Met Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Val Ile Gly Arg
260 265 270
CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC TGT 864
CAA GAA GCA TAT GGT
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Cys
Gln Glu Ala Tyr Gly
7S 275 280 285
GTG AGC GTC ATT GTG GGA GTA CCT CCT GAT TCC 912
CAA AAT CTC TCT ATG
Val Ser Val Ile Val Gly Val Pro Pro Asp Ser
Gln Asn Leu Ser Met
290 295 300
80
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG 960
AAA GGA GCT ATT TTT
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
Lys Gly Ala Ile Phe
305 310 315
SUBSTITUTE SHEET (RULE 26)
~ T
CA 02290074 1999-11-12
WO 98/51802 PCTNS98/09627
53
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA CTT GTG GCC GAT TTT 1008
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe
320 325 330 335
S ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC ACC CAT GTT TTA CCT 1056
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro
340 345 350
TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT CGC TCT GGA GAG AGT 1104
IO Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser
355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
Ile Arg Thr Ile Leu Thr Phe
15 370
(2) INFORMATION FOR SEQ ID N0:18:
2 O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
25 (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
3 O Sei Thr Ala Gly Lys Val Ile Lys Cys Lys Ala Ala Val Leu Trp Glu
15
Glu Lys Lys Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys
25 30
35 Ala His Glu Val Arg Ile Lys Met Val Ala Thr Gly Ile Cys Arg Ser
35 40 45
Asp Asp His Val Val Ser Gly Thr Leu Val Thr Pro Leu Pro Val Ile
4O 50 55 60
Ala Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val
65 70 75 BO
Thr Thr Val Arg Pro Gly Asp Lys Val Ile Pro Leu Phe Thr Pro Gln
45 85 9p g5
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Leu Cys Leu
100 105 110
5 O Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
55 130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
6O 165 170 175
Ser Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
180 185 190
65 Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile
7O 210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys G1u Va1 Gly Ala Thr Glu Cys
225 230 235 240
Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu
75 z4s z5o zss
Met Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Val Ile Gly Arg Leu
260 265 270
gO Asp Thr Met Val Thr Ala Leu Ser Cys Cys Gln Glu Ala Tyr Gly Val
275 280 285
Ser Val Ile Val Gly Val Pro Pro Asp Ser Gln Asn Leu Ser Met Asn
290 295 300
SUBSTITUTE SHEET (RULE 26)
i i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
S4
ProMetLeuLeuLeuSerGlyArgThrTrpLysGly IlePheGly
Ala
305 310 315 320
S GlyPheLysSerLysAspSerValProLysLeuVal AspPheMet
Ala
325 330 335
AlaLysLysPheAlaLeuAspProLeuIleThrHis LeuProPhe
Val
340 345 350
GluLysIleAsnGluGlyPheAspLeuLeuArgSer GluSerIle
Gly
355 360 365
ArgThrIleLeuThrPhe
1 370
S
(2) N0:19:
INFORMATION
FOR
SEQ
ID
Z (i)SEQUENCE
O CHARACTERISTICS:
(A) pairs
LENGTH:
1128
base
(B) nucleic acid
TYPE:
(C) double
STRANDEDNESS:
(D) linear
TOPOLOGY:
ZS
(ii)MOLECULE DNA(genomi c)
TYPE:
(Xi)SEQUENCE SEQID :
DESCRIPTION: N0:19
3
O
ATG AGCACAGCA AAAGTAATAAAATGCAAAGCGGCTGTGCTGTGG 48
GGA
SerThrAla LysValIleLysCysLysAlaAlaValLeuTrp
Gly
1 5 10 15
3 GAG GAAAAGAAA TTTTCCATCGAGGAGGTGGAGGTTGCACCCCCG 96
S CCA
Glu GluLysLys PheSerIleGluGluValGluValAlaProPro
Pro
20 25 30
AAG GCCCATGAA CGTATAAAGATGGTGNNNACAGGAATTTGTCGC 144
GTC
40 Lys AlaHisGlu ArgIleLysMetValAlaThrGlyIleCysArg
Val
35 40 45
TCA GATGACCAC GTTAGTGGAACCCTTGTCACACCTCTTCCTGTG 192
NNN
Ser AspAspHis ValSerGlyThrLeuValThrProLeuProVal
Val
4S 50 55 60
ATC GCAGGCCAT GCAGCGGGCATTGTGGAGNNNATTGGAGAAGGC 240
GAG
Ile AlaGlyHis AlaAlaGlyIleValGluXaaIleGlyGluGly
Glu
6s 70 75
SO
GTC ACTACAGTA CCAGGTGATAAAGTCATCCCACTCTTTNNNCCC 288
AGA
Val ThrThrVal ProGlyAspLysValIleProLeuPheXaaPro
Arg
80 85 90 95
S CAG TGTGGAAAA AGGGTTTGTAAGCACCCTGAAGGCAACNNNTGC 336
S TGC
Gln CysGlyLys ArgValCysLysHisProGluGlyAsnXaaCys
Cys
100 105 110
TTG AAAAATGAT AGCATGCCTCGGGGAACCATGCAGGATGGTACC 384
CTG
60 Leu LysAsnAsp SerMetProArgGlyThrMetGlnAspGlyThr
Leu
115 120 125
AGC AGGTTCACC AGAGGGAAGCCCATCCACCACTTCCTTGGCACC 432
TGC
Ser ArgPheThr ArgGlyLysProIleHisHisPheLeuGlyThr
Cys
6S 130 135 140
AGC ACCTTCTCC TACACCGTGGTGGACGAGATCTCAGTGGCCAAG 4B0
CAG
Ser ThrPheSer TyrThrValValAspGluIleSerValAlaLys
Gln
145 150 155
7O
ATC GATGCGGCC CCGCTGGAGAAAGTCTGTCTCATTGGCTGTGGA 528
TCA
Ile AspAlaAla ProLeuGluLysValCysLeuIleGlyCysGly
Ser
160 165 170 175
7S TTT NNNACTGGT GGGTCTGCAGTCAAGGTTGCCAAGGTCACCCAG 576
TAT
Phe XaaThrGly GlySerAlaValLysValAlaLysValThrGln
Tyr
180 185 190
GGC TCCACCTGT GTGTTTGGCCTTGGAGGAGTGGGCCTGTCTGTT 624
GCC
g0 Gly SerThrCys ValPheGlyLeuGlyGlyValGlyLeuSerVal
Ala
195 200 205
ATC ATGGGCTGT GCAGCCGGAGCGGCCAGGATCATTGGGGTGGAC 672
AAA
Ile MetGlyCys AlaAlaGlyAlaAlaArgIleIleGlyValAsp
Lys
SUBSTITUTE SHEET (RULE 26)
~r i t
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
SS
210 215 220
ATC AAC AAA GAC AAG TTT GCA AAG GCC AAA GAA 720
GTG GGT GCC ACT GAG
Ile Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu
S Val Gly Ala Thr Glu
2
25 230 235
TGT GTC AAC CCT CAG GAC TAC AAG AAA CCC ATC 768
CAG GAG GTG CTG ACA
Cys Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile
Gln Glu Val Leu Thr
l0 240 245 250 255
GAA NNN AGC AAT GGA GGT GTG GAT TTT TCC TTT 816
GAA NNN ATT GGT CGG
Glu Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe
Glu Xaa Ile Gly Arg
260 265 270
IS CTC GAC ACT ATG GTG ACT GCC TTG TCA TGC NNN 864
CAA GAA GCA TAT GGT
Leu Asp Thr Met Val Thr Ala Leu Ser Cys Xaa
Gln Glu Ala Tyr Gly
275 280 285
GTG AGC GTC ATT NNN GGA GTA CCT CCT NNN TCC 912
2 CAA AAT CTC TCT ATG
0
Val Ser Val Ile Xaa Gly Val Pro Pro Xaa Ser
Gln Asn Leu Ser Met
290 295 300
AAT CCT ATG TTG CTA CTG AGT GGA CGT ACC TGG 960
AAA GGA GCT ATT TTT
Asn Pro Met Leu Leu Leu Ser Gly Arg Thr Trp
ZS Lys Gly Ala Ile Phe
3
0
310 315
GGC GGT TTT AAG AGT AAA GAT TCT GTC CCC AAA 1008
CTT GTG GCC GAT TTT
Gly Gly Phe Lys Ser Lys Asp Ser Val Pro Lys
Leu Val Ala Asp Phe
3 320 325 330 335
O
ATG GCT AAA AAG TTT GCA CTG GAT CCT TTA ATC 1056
ACC CAT GTT TTA CCT
Met Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile
Thr His Val Leu Pro
340 345 350
3 TTT GAA AAA ATA AAT GAA GGA TTT GAC CTG CTT 1104
S CGC TCT GGA GAG AGT
Phe Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu
Arg Ser Gly Glu Ser
355 360 365
ATC CGT ACC ATC CTG ACG TTT TGA 1128
40
Ile Arg Thr Ile Leu Thr Phe
370
(2)INFORMATION SEQ ID N0:20:
FOR
4S
(i)SEQUENCECHARACTERISTICS:
(A) adds
LENGTH:
374
amino
(B)
TYPE:
amino
acid
(D)
TOPOLOGY:
linear
5
O
( ii)MOLECULETYPE: protein
(xi) SEQUENCEDESCRIPTION: N0:20:
SEQ ID
S SerThrAlaGly Val Ile LysA1aAlaValLeuTrpGlu
S Lys Lys Cys
1 S 10 IS
GluLysLysPro Ser Ile ValGluValAlaProProLys
Phe Glu Glu
20 25 30
60
AlaHisGluVal Ile Lys XaaThrGlyIleCysArgSer
Arg Met Val
35 40 45
AspAspHisXaa Ser Gly ValThrProLeuProValIle
Val Thr Leu
6S So Ss 60
AlaGlyHisGlu Ala Gly GluXaaIleGlyGluGlyVal
Ala Ile Val
65 70 75 80
70 ThrThrValArg Gly Asp IleProLeuPheXaaproGln
Pro Lys Val
85 90 95
Cys Gly Lys Cys Arg Val Cys Lys His Pro Glu Gly Asn Xaa Cys Leu
100 105 110
7S
Lys Asn Asp Leu Ser Met Pro Arg Gly Thr Met Gln Asp Gly Thr Ser
115 120 125
Arg Phe Thr Cys Arg Gly Lys Pro Ile His His Phe Leu Gly Thr Ser
g0 130 135 140
Thr Phe Ser Gln Tyr Thr Val Val Asp Glu Ile Ser Val Ala Lys Ile
145 150 155 160
SUBSTITUTE SHEET (RULE 26)
i i
CA 02290074 1999-11-12
WO 98/51802 PCT/US98/09627
56
Asp Ala Ala Ser Pro Leu Glu Lys Val Cys Leu Ile Gly Cys Gly Phe
165 170 175
Xaa Thr Gly Tyr Gly Ser Ala Val Lys Val Ala Lys Val Thr Gln Gly
180 185 190
Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Ile
195 200 205
l~ Met Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Ile
210 215 220
Asn Lys Asp Lys Phe Ala Lys Ala Lys Glu Val Gly Ala Thr Glu Cys
225 230 235 240
15 Val Asn Pro Gln Asp Tyr Lys Lys Pro Ile Gln Glu Val Leu Thr Glu
245 250 255
Xaa Ser Asn Gly Gly Val Asp Phe Ser Phe Glu Xaa Ile Gly Arg Leu
250 265 270
Asp Thr Met Val Thr Ala Leu Ser Cys Xaa Gln Glu Ala Tyr Gly Val
275 280 285
S Ser Val Ile Xaa Gly Val Pro Pro Xaa Ser Gln Asn Leu Ser Met Asn
290 295 300
Pro Met Leu Leu Leu Ser Gly Arg Thr Trp Lys Gly Ala Ile Phe Gly
305 310 315 320
3 O Gly Phe Lys Ser Lys Asp Ser Val Pro Lys Leu Val Ala Asp Phe Met
325 330 335
3 5 Ala Lys Lys Phe Ala Leu Asp Pro Leu Ile Thr His Val Leu Pro Phe
340 345 350
Glu Lys Ile Asn Glu Gly Phe Asp Leu Leu Arg Ser Gly Glu Ser Ile
355 360 365
Arg Thr Ile Leu Thr Phe
370
4S (2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: single
S ~ (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
SS (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
CCCCGAATTC TCAAAACGTC AGGATGGTAC G 31
6O (2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(7S (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
70 CCCCTCTAGA ATAAATGAGC ACAGCAGGAA AAGTAATAAA ATGC 44
SUBSTITUTE SHEET (RULE 26)
