Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SMB
Meth,Qd for Produdn~nd Idgntr~~ng New
H,_ydr2,(ase~ Ha~(,i,~g Improved Prod rtie~
The present invention relates to a process for the preparation and
identification of hydrolase mutants having improved properties with
respect to stereo- or regioselectivity, catalytic activity or stability in
chemical reactions.
P ri r a
Hydrolases are among the most wide-spread enzymes in organic syn-
thesis. As a subgroup of the hydrolases, esterases and lipases, in
particular, catalyze a wide variety of reactions, such as the hydrolysis of
carboxylic acid esters, yr the synthesis of esters or transesterifications in
organic solvents. Due to their high stereoselectivity, stability and their
being readily available, they are interesting for numerous industrial
processes. Thus, for example, lipases have been industrially employed
for the optical resolution of chiral alcohols, acids or amines, for the
preparation of optically pure medicaments, natural substances, plant
protective agents or high-grade fats and oils (K. Faber, 8iotransforma-
tions in Organic Chemistry, Springer-Verlag, Berlin, 2nd Ed. 1995).
Nevertheless, the enantioselectivity of a lipase or esterase with
respect to a given substrate cannot be predicted with certainty, and in
many cases, the reactions proceed with only moderate optical yields.
Therefore, there is a need for a process for the preparation of hydro-
lases which enables a well-aimed optimization of enantioselectivity
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with respect to a desired product and the special process conditions,
such as temperature and solvent. Although effects on the enantiose-
lectivity of iipases could be studied using the molecular-biological
method of in vitro mutagenesis, which is customary today (K. Hult, M.
Holmquist, M. Martinelle, European Symposium on Biocatalysis, Graz,
1993, Abstracts, L-4), an optimization with respect to a particular
substrate which would have led to an enzyme useful in organic syn-
thesis could not be achieved.
The most important possible applications of genetic engineering include
protein desig, wherein mutations are introduced base-specifically into
the gene sequence of the corresponding protein based vn known
structural data using in vitro mutagenesis. By selectively substituting
amino acids, enzymes having improved catalytical activity or stability
could already be prepared in this way {A. Shaw, R. Bott, Current Opinion
in Structural Biology, 1996, 6, 546). This technique, the so-called
oligonucleotide-directed or site-directed mutagenesis, is based on the
substitution of a short sequence segment of the gene coding for the
naturally occurring enzyme (wild type) by a synthetically mutagenized
oligonucleotide. Subsequent expression of the gene results in a n
enzyme mutant which may have advantageous properties. In a
method derived therefrom, the so-called cassette mutagenesis, oli-
gonucleotides with partially randomized sequences are used. This
provides a library of mutants of a limited size, which can then be tested
with respect to its properties.
Despite of advantages of these established methods,they are
the
hardly suitablefor the stepwise optimization enzyme or for
of an the
generation enzymes having novel properties. The factthat our
of
understandingof the laws governing protein and the structur'e-
folding
function relationship of proteins is still incompleteis the
main
reason
for the failingof many projects in the field
of the so-called rational
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3 -
protein design. In addition, a stepwise optimization process according
to the classical method is relatively labor-consuming and does not
ensure a significant improvement of the enzyme properties per se.
More recently, novel molecular-biological methods of mutagenesis have
been described (D.W. Leung, E. Chen, D.V. Goeddel, Technique, 19$9,
I, 11, and W.P.C. Stemmer, A. Crameri, PCT WO 95/22625) which are
based on the polymerase chain reaction known from the literature (R.K.
Saiki, S.J. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, N. Arn-
heim, Science, 1985, 230, I3S0). Instead of site-directed mutagenesis,
these methods employ combinatorial methods for the generation of
extensive mutant libraries which are subsequently screened for mu-
tants having positive properties using suitable screening methods. This
mimics the naturally occurring evolutive processes of replication and
recombination, mutation and selection on a molecular level. This
method, described as in ultra evolution for directed evolution), has
already proven useful in some cases as a suitable method for obtaining
new biocatalysts (W.P.C. Stemmer, Nature, 1994, 370, 389, and F.H.
Arnold, Chemical Engineering Science, 1996, 51, 5091).
In spite of the progress made in this field, this method cannot yet be
generally transferred to all classes of enzymes, since suitable test
methods for identifying mutants with positive properties are lacking in
most cases. Such methods are a sine qua non, however, in view of the
large number of mutated enzyme variants to be expected in the
production of combinatorial mutant libraries. Especially in the case of
the lipases which are interesting for industrial processes, the produc-
tion of mutants with improved stereoselectivity by the methods of in
vitro evolution has not been successful to date, because an efficient
screening method for enantioselectivity testing still does not exist. The
classical method for determining the enantioselectivity of a lipase- or
esterase-catalyzed reaction is based on the separation of the reaction
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products and educts by iipuid or gas chromatography using chirally
modified stationary phases. However, due to the enormous number of
samples to be processed in the screening of extensive mutant Libraries,
this method is unsuit~Jble since chromatographical separations with
chirally modified columns are time-consuming, being only capable of
sepuential processing. Another as yet unsolved problem is the diffi-
culty, frequently to observe, of expressing functional lipases or es-
terases in host organisms with a sufficiently high activity yield. How-
ever, this is indispensable to a high-performance screening system
since too low enzyme activities are difficult to detect in the determina-
tion of enantivselectivity due to the limited sensitivity of a test system.
Obigct~f the inyention
Therefore, it has been the object of the present invention to provide a
simple process for the preparation of mutated hydrolases, especially
iipases or esterases, having improved stereo- or regioselectivity,
catalytic activity and stability towards particular substrates (e.g.,
carboxylic acids, alcohols, amines, or their derivatives), which process
additionally enables a rapid identification of positive mutants from
extensive mutant libraries, and the use of the enzymes thus prepared
in the optical resolution of chiral alcohols, acids and amines, and their
derivatives.
Descri~t'on of ~,he inygntion
The present Invention relates to
(1) a profess for the prepacatlvn and identificaflon of hydrole~sa mutants
having
improved properric~s with respect to stereo- or regioseiectiv;ty, catalytic
activity or
stability, charattenxed in that
a) a starting hydrofase gene is mutagenized ~y a modified pofymel-ase rttoin
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4a
reaction (PCR), wherein the mutation rate and total number of mutations in the
amplified DNA is adjusted by adjusting the ~nnCentrations of Mgz*, MnZ+ and of
the
deoxynucleOttdes and by adjusting tht number of cycles; and/or
b) one or more starting hydrolase genes, one or mor-c hyd~olase genes mutated
acCVrdtng to step a), or mixtures of one or more starting hydrolese genes and
one
or mere l~ydroiase genes mutated according to step a) are mutagenized by
enxymaticbity fragmenting said genes, followed by enzymatic rer~ssembly of the
rtragrrtenzs produced to give complete recombinant hydrolase genes;
c) t>~e mutated hydrolase genes obkained according to step a) or b) are
transformed Into a host organism; and
d) hydrolast mutants having improved properties, expressed by transformants
obtained in step c), are identified by a test method;
(2) a hydrolae mutant obtainable by the process defined under (1):
(3) a oNA sequence e:oding for the hydrolase mutant defined uneer (z);
{4) a vector comprising the DNA sequence defined under (3);
{5) a CranSfvrmant comprising a DNA sequence as det5ned under (3) and/or a
vector as defined under (4);
(8) a process for the preparation of hydralas~ mutants hawir~~ in7praved
properties, cornprlsing culturing a transfvrmant bccording to (5); and
(7) a mesthod for testing catalysts for stereo- or regioselectivity, wherein
CquBl
amounts of the catalyst are added to a test substrate and to the pure stereo-
or
reflioisomers of the test substrate, provided with a chrvmvphorous group which
Causes a spectrometrically determinable change of absorption or emtsston upon
cleavage by the catalyst, in separate test vessels, and the stereo- or
regioselectivf~Cy Is defiermined from the ratio of the linear init;at reason
rates
obtained.
As a rule, the preparation of the new biocatalysts starts wirh the isolation
of a
lip~rse or esterase gene from the organism of origin. This may be any
filCrobial,
plant and animal organism which is the carrier oP a lipase or esterase gene.
The
isolation of the gene can be effected according to the mothods known Pram the
literature (J. Sambrook, E.F. Fritsch, 'f. Maniatts, MOleCUlar Clanlng: A
Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1989, New York). Usually, the
genwnic bNA Is rtragmented using restriction endanuclee~ses, and the gene
fragments obtained are cloned in a host organism i;e.g., ~ co~~')- Then, using
otigonucieotides with 5equenCQ homology ic'o a segment of the IipaSG or
esterase
gene, the gene is identified witt,in the gene libr5ry tn hybridization
experiments,
followed by isolation thereof.
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-
Surprisingly, it has been found according to the invention that naturally
occurring hydrolase genes can be mutagenized by a modified polym-
erase chain reaction (PCR), changing certain reaction parameters, to
obtain an extensive mutant library which can be screened for mutants
having improved enantioselectivity using a novel test method.
The novelty of the process resides in that an extensive randomized
mutant library can be established, starting with a naturally occurring
lipase or esterase gene (the so-called wild type gene), using a modi-
fied PCR {hereinafter referred to as mutagenizing PCR). It has been
found that the mutation rate during the PCR can be adjusted in a well-
aimed manner by changing the components of the PCR: The number of
mutations in the lipase gene in question (the mutation rate) can be
controlled by varying the concentrations of Mgz+ and/or of the deoxy-
oligonucleotides and/or the addition of Mn2+ ions. preferably, the
following concentrations are used depending on the DNA polymerase
employed:
Mg2+: I.5 mM - 8.0 mM
dNTP:0.05mM-I.OmM
Mnz+: 0.0 mM - 3.0 mM
In addition, it has been found that the number of cycles in the PCR
correlates with the number of mutations: the higher the selected
number of cycles, the higher is the total number of mutations. By
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- 6 -
means of this parameter, the diversity of the mutant library can be
adjusted.
For' determining the mutation rate, the pufified PCR products are
sequenced. The mutation rate can be determined by comparing the
sequences obtained with the sequence of the wild type gene.
Table 1 shows the mutation rate as a function of the concentration of
the above mentioned components of the PCR in the amplification of the
lipase gene from P, aeruginosa (IipA).
T_ able 1
Exp. Mg'+ Mn'+ dATP/ dTTP/ Mutation rate
(mM) (mM) dGTP dCTp (mutations/
(mM) (mM) 1000 bpl~)
1 ~ 6.1 ~ - ~ 0.2 0.2 1-
2 ~ 7.0 l 0.5 I 0,2 ( I.0 ~ 15-20
1~ by = base pairs
From the sequencing results, it can further be seen that the transition
and transversion types of mutation occur in about the same statistical
frequency. In contrast, deletions and insertions are rarely observed. In
addition, the mutations are uniformly distributed over the entire lipase
gene. Thus, a mutant library with statistically uniformly distributed
mutations can be produced by the method described. A mutation rate
of 1-2 mutations/hydrofase gene has proven advantageous. Thereby,
it is prevented that a negative mutation will mask a mutation with a
positive effect, as would be the case if several mutations occurred per
one hydrolase gene. In order to obtain a complete mutant library, each
with one amino acid substitution per enzyme molecule, 5415 mutants
must theoretically be generated in a lipase consisting of 285 amino
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acids {here: lipase from P. aeruginosa). This value results from the
following formula:
N = 19 x M x 2$5! / [(2$5 - M)! x M!]
with N = number of mutants, and M = number of amino acid substitu-
tions per one lipase molecule. According to the invention, it could be
surprisingly shown that positive mutants are found in even substan-
tially smaller sized libraries, a mutation rate of 1-2 having been em-
ployed.
The mutated lipase or esterase genes obtained by the process de-
scribed are ligated into a suitable expression vector and then trans-
formed into a host organism, e.g., E. coil. Then, the transformed cells
are plated on agar plates and cultured. rf the expression rate is
suffrciently high, the colonies obtained can be transferred to microtitra-
tion plates provided with a liquid medium and, after growth has
started, can be directly employed in a screening test. In the case
where only little enzyme is formed in the expression of the lipase gene
or the gene product is not correctly folded in the host organism used
(inclusion bodies) or incompletely secreted into the culture medium, it
will be advantageous to reclone the mutated genes in another host
organism, preferably the original organism.
In order to obtain sufficiently high enzyme activities, the individual
bacterial clones which contain a mutated lipase or esterase gene are
transferred from the agar plates into the wells of commercially avail-
able microtitration plates and cultured in liquid medium. Preferably,
microtitration plates having 9fi wells per plate are employed. The
growth of the bacteria can be monitored by measuring the cell density
(~~60o value). It is advantageous to inoculate a second microtitrativn
plate in parallel in this way in order to have a reference for the later
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identification of positive clones. After the growth of the bacteria glyc-
erol is conveniently added to the reference plate, which is then stored
at -80 °C until used for identification. If the bacteria are secreting
the
enzyme into the extracellular space (as with the lipase from P. aerugi-
nosa), the cells in the microtitration plates are centrifuged off, and the
supernatant with the lipase or esterase activity is used for the
screening test. In the case where the bacteria (e.g., E. coh~ accumulate
the enzyme in the periplasm, a cell wall lysis must be preliminarily
done, wherein methods known from the literature, such as lysozyme
treatment, can be used.
By culturing the corresponding clones from the reference plate, suffi-
cient plasmid DNA can be isolated which can be used for the charac-
terization of the mutated lipase or esterase gene. The mutations are
localized within the gene by sequencing, One advantage of the inven-
tion is the fact that the mutated gene in a positive clone can be further
optimized with respect to its properties in further mutation cycles by
the process described, even without knowing the exact position of the
mutations. Thus, the isolated lipase or esterase gene is again used in
a pCR modified according to the above stated conditions {mutagenizing
PCR). This procedure may be repeated until the properties of the lipase
or esterase mutant meet the requirements of the stereoselective
reaction.
For a further optimization of the identified positive mutants, the prod
ess described can be extensive in that the DNA of several positive
mutants is first fragmented and then can be reassembled into func-
tional lipase or esterase genes in a combinatorial process according to
W.P.C. Stemmer (Nature, 1994, 370, 389). The thus obtained in vitro
recombinant library is subsequently expressed, and the recombinant
gene products are examined for improved enantioselectivity using the
test methods according to the invention. The advantage of this method
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is that the positive properties of different lipase or esterase mutants
may be added in one new recombinant gene due to the recombination,
which eventually may result in a further improvement of the lipase or
esterase. The course of the method described is as follows:
Using the enzyme DNase I (e.g., from bovine pancreas), the lipase or
esterase genes are first cleaved into fragments having a preferably
length of between 25 by and 100 bp. The size of the fragments can b a
checked by separating them by means of agarose electrophoresis and
comparing with corresponding DNA length markers. The DNA fragments
thus obtained are purified to free them from adhering DNase. The in
vitro recombination is performed under the conditions of a conventional
PCR, but without adding any PCR primers. In analogy with conventional
PCR, one cycle is comprised of three steps: a) denaturing, b) annealing
and c) elongation. During annealing, hybridization occurs of sequence-
homologous fragments which may be derived from different mutated
lipase or esterase genes. In the subsequCnt elongation step, the
strands are completed by the DNA polymerase so that new recombi-
nant lipase genes are eventually obtained. The optimum number of
cycles is determined in a preliminary experiment. Thus, after every 5
cycles, a small sample of the reaction mixture is separated by agarose
gel electrophoresis to determine the cycle in which the maximum of the
size distribution of the recombinants in the range of the size of the
enzyme gene. A number of cycles of between 30 and 45 is preferably
selected. The band obtained in the agarose gel which corresponds in
size to the lipase or esterase gene is purified and amplified by a
conventional PCR. The PCR product is purified and, Following ligation
into a suitable vector (plasmld), transformed into ~ coli. As already
discussed in the paragraph dealing with mutagenrzing PCR, it may be
required to reclone in another host organism if the lipase activity
should be too low after expression in E. coli. The recombinants ob-
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-~.a-
tained are grown in microtitration plates for the test for enantioselec~
tivity.
In a variant of the invention, the described methods of mutagenizing
PAR and in vitro recombination for the production of mutant or recombi-
nant libraries can be performed successively or repeated in any order
and frequency desired in order to optimize the enantioselectivity of the
lipase or esterase. Preferably, at least one mutation cycle is performed
in the beginning using mutagenizing PCR. This may then be followed by
an in vitro recombination cycle, wherein the best positive mutant clones
are respectively employed. By monitoring the enantioselectivity of the
enzyme mutants obtained, the optimization process can be followed.
In another variant of the invention, positive lipase or esterase mutants
identified by the screening of mutant or recombinant libraries can be
further optimized using classical directed mutagenesis or cassette
mutagenesis. Thus, the mutation in the lipase or esterase gene is first
localized by sequencing. This gene is subsequently again mutagenized
by means of "wobbled" primers at the codons coding for positive
mutants. The thus obtained mutant library of a limited size can then be
expressed and screened for improved enantioseiectivity.
Positive lipase or esterase mutants identified by the screening of
mutant or recombinant libraries can be further optimized using site-
directed saturation mutagenesis. Thus, the positive mutation in the
lipase or esterase gene is first localized by sequencing. Then, using
any method of site-directed rnutagenesis which allows for the ex-
change of multiple bases, this gene is changed in such a way that ail
possible codons are formed at the site of the gene which codes for the
position to be optimized. This provides a library of mutants of a limited
size in which mutants the amino acid originally present in the amino
acid position to be optimized has been replaced by the remaining 19
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ID:
amino acids. The thus obtained mutant library of a limited size can then
be expressed and screened for improved enantioselectivity.
In a variant of the method described, the lipase or esterase gene of
the wild type enzyme is employed for in vitro recombination together
with the positive mutants found. This can result in backcrossings in
which mutations having neutral or negative properties can be elimi-
nated. Following expression, the recombinant library obtained can be
examined for improved enantioselectivity.
in another variant of the method described, hydrolase genes from
different organisms are employed for in vitro recombination, provided
they possess sufficient sequence homology with the originally em-
ployed hydrolase gene.
In a variant of the method, the in vitro recombination is performed
under the conditions of the modified PCR described. Thus, the concen-
trations of the Mgz' or Mn2+ ions and of the deoxynucleotides (dNTPs)
are changed to adjust the mutation rate during in vitro recombination
in a well-aimed manner.
The invention further relates to test methods which allow for the
identification of enzyme mutants having Improved stereoselectivity or
regioselectivity from extensive mutant libraries. Thus, after centrifuging
off the bacterial cells, two alipuvts of the enzyme-containing super-
natant are transferred to adjacent wells of a new microtitration plate.
After addition of the two enantiomeric pure substrates in the two
wells, respectively, the activity of the lipase or esterase is determined
by spectrophotometry. The measurements are performed in a commer-
cially available spectral photometer for microtitration plates. This allows
for a high sample throughput. The selection of the substrate depends
on the type of chiral compound for which optimization of the lipase or
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- 12 -
esterase is to be effected. The method is particularly suitable for chiral
carboxylic acids, alcohols and amines.
In the case of chiral carboxylic acids or chiral COOH-functions( com-
pounds, the two corresponding p-nitrophenyl esters of the (R)- and
(5)-acids are employed as test substrates. Formula 1 shows the
principle of the test method wherein R represents any organic residue
having at least one asymmetric center.
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- 13 -
Form la
Scheme of the test method for stereoselectivity for chiral carboxylic
acids or COOH-functional compounds
Reaction 1:
.~"w..._ Y
ut~ i
t H'~ '._ _.'" , .-"~."w r~'~ . ~: .~ I,~.
A ,7 Ft U!i ;C~-' ,w,,~.r~
lR~rtsnGOr~
Reaction 2:
MCS~. n ,~ .,~., "'ny
,, r~:~ ~"
~t ~ ~'
i~l~Eryar~ya~t~er
Due to the high absorbance of the p-nitrophenolate anion released in
the hydrolase-catalyzed ester hydrolysis (a,max = 405 nm,
Emax = 14,000), a highly sensitive test method results by which a n
activity determination can be performed even for low substrate concen-
trations. The enantioselectivity of the hydrolase mutants can be
determined with sufficient accuracy from the quotient of the hydrolysis
rates Vapp(R) and Vapp(S) for the (R)- and the (S)-ester, respectively.
Since both test reactions contain only one enantiomer (either the R- or
the S-ester), the absence of a competing reaction with the other
enantiomer must be taken into account when the enanti0selectivity is
determined. Although this kinetic effect may lead to the calculation ,of
inaccurate enantioselectivities, it has been found that the apparent
enantloseiectivities obtained by the presented method (Eapp) are
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- 14 -
sufficiently telling with respect to the enantioselectivity of the mutated
lipases. Eapp is obtained as Vapp(R)/Vapp(s)~ Another advantage is the
simple pertormance and good reproducibility of the test, which is also
suitable for screening with a high sample throughput.
In the case of chiral alcohols or chiral OH-functional compounds, fatty
acid esters of the two optically pure alcohols are employed in the test
for stereoselectivity. The chain length of the fatty acids is within a
range of from C2 to Cis. As the alcohol component, primary, secondary
and tertiary alcohols and their derivatives having at least one asym-
metric center can be used. Solutions of the esters of the (R)- and (S)-
alcohols are hydrolysed with culture supernatants of the hydrolase
mutants in adjacent wells of a microtitration plate. The hydrolysis rates
Vapp~R~ and Vapp~s~ for the (R)- and the (S)-ester, respectively, are a
measure of the enantioselectivity of the enzyme mutant examined.
Detection is effected through a coupled enzyme reaction (H.U. Berg
meyer, Grundlagen der enzymatischen Analyse, Verlag Chemie, Wein-
heim, 1977) in which the continuous release of the fatty acid is moni-
tored. The dye produced is assayed by colorimetry at 546 nm
(c = 19.3 1~mmol'' ~cm-1). The concentrations of the enzymes, cofactors
and coenzymes of auxiliary reactions Z and 3 (see Formula 2) and of
the indicator reaction 4 must be selected in such a way that the lipase-
or esterase-catalyzed reaction to be determined is rate-determining.
The quotient of the hydrolysis rates for the (R)- and the (S)-ester,
respectively, corresponds to the apparent enantioselectivity (Eaap). In
one variant, the fatty acid amides of chiral amines or NHz~ or IVHR-
functional compounds are employed instead of the optically pure
esters, Formula Z shows the scheme of the test system.
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- 15 -
F~~mula 2
Scheme of the test method for stereoselectivity for chiral alcohols; R
represents any organic residue having at least one asymmetric center;
abbreviations: CoA {coenzyme A), ATP (adenosine-5'-triphosphate),
AMP (adenosine-5'-monophosphate)
0
0
1. ) R w H~1~ ~. + ~
n yo~H
free fatty acid
2. ) free fatty acid + CoA + ATP ,~~ S~ aryl-CoA + AMP + pyrophosphate
~4Gtr!-GaA, O.~art>fas~e
3, ) acyl-CoA + O: --..~,.~:........~.~. enpyl-CoA + HzOz
~~~I~tld~lss
4, ) HxOz + a-aminoantipyrine + 2,4,6-tribromo-3-hydroxybenzoic acid ,-~---
.red dys +
2 H20 + HBr
In a variant of the method, the corresponding esters and amides of
succinic acid can be employed instead of the fatty acid esters or
amides. The latter have the advantage, over the fatty acids, of being
more soluble in aqueous solutions or aqueous-organic solvents. The
measurement is performed by UV spectrometry at 34Q nm
(~ = 6.3 l~mmol-l~cm-1). In this test method too, it has to be taken care
that the hydrolase-catalyzed reaction 1 be rate~determining. The
quotient of the hydrolysis rates Vapp~R) and Vapp(S) for the (R)- and the
(S)-ester, respectively, corresponds to the apparent enantioselectivity
(Eapp). In one variant, the fatty acid amides of chiral amines are em-
ployed instead of the optically pure esters. Both primary and secondary
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16 -
amines may be employed as the amine component. The scheme of the
test system is represented in Formula 3.
Formula 3
Scheme of the test method for stereoselectivity for chiral aicohols; R
represents any organic residue having at least one asymmetric center;
abbreviations: CoA (coenzyme A), ITP (inosine-5'-triphosphate), IDP
(inosine-5'-diphosphate), NADH/NAD+ (reduced/oxidized nicvtinamide
adenine dinucleotide)
1. ) Ht~OC.,~,,r~,~,~~~JR + Hz0 L ~9~ ""'.,.~..~,., . H'!N + R-OH
free succinic acid
Sucdr,~rl-GSA. sy~ntt~el~
2, ) Succinic acid + ITP + CQA '~~" IDP + succinyl-CoA + phosphate
Py~c~mle knish
3 , ) IOP + phosphaeno( pyruvate ITP + pyruvate
LSC~arig ~~ahydroga~sse
g., ) Pyruvate + NADH + H''' L-lactate +NAD'
The test for the identification of hydro(ase mutants having improved
stereoseiectivity may further be performed in such a way that both
stereoisomers are contained in the test reaction. Thus, the separated
measurements of the (R)- and (S)-enantiomers can be dispensed with.
The test principle starts with binding a racemic mixture of the chiral
substrate to a solid phase. Through an ester or amide linkage to this
ch(ral compound, a radioactively labeled organic residue is bound. Two
cases can be distinguished:
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- 17
a) Solid-phase bound chiral carboxylic acid: the carboxy function is
esterified with a radioactively labeled alcohol.
b) Solid-phase bound chiral alcohol or chiral amine, or OH- or NHz-
functional (or NHR-functional) compounds: the hydroxy or amine
function is labeled with a radioactively labeled carboxylic acid.
It is critical that the two enantiomers of the racemic mixture bound to
the solid phase be labeled with different isotopes, Preferably, 3H- and
laC-labeled compounds are used. As the solid phase, all usual organic
functionalized polymers as well as inorganic functionalized supports
can be employed. Preferably, solid phases based on polystyrene and
silica gel supports are employed. The chiral radioactively labeled
compounds are then bound to the solid phase wherein the coupling to
the solid phase must be adapted to the chemical nature of the chiral
substrate. Formula 4 shows the scheme of the modified solid phase
and the principle of the test method.
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- 18 -
Formula 4
Scheme of the solid-phase screening test for stereoselectivity with a
dual radioactively labeled substrate; X = 0, NH; R is a radioactively
labeled organic residue
Q
t ~_
~ ~ s-~ .~ x~~ ~o I~
f 1
~ro~ia
H~~ O
I~~-C~-~ l C1
X' '14- -R
Approximately equal amounts of the thus modified support can be
dispensed to small reaction vessels (e.g., the wells of microtitration
plates) and then admixed with the culture supernatants of the hydro-
(ase mutants. In the subsequent reaction, the radioactively labeled
components {carboxylic acid or alcohol) are hydrolysed from the solid
phase and released into the liquid medium. An aliquote of the medium
is then removed and examined for the amount of radioactivity in a
scintillation counter. From the ratio between the two different isotopes,
the enantiomeric excess and the conversion of the reaction and thus
the enantioselectivity of the mutated esterase or lipase can be calcu~
lated. Sy using regioisomeric test compounds, the tests described can
also be used for the identification of hydrolase mutants having im-
proved regioselectivity. Instead of hydrolase mutants, other catalysts
may also be employed for determining the stereo- or regioselectivity:
The test for enantioselectivity of the hydrolase mutants prepared by
the process described may also be performed by a capillary-
electrophoretical separation using chirally modified capillaries which
allow for a direct separation of the enantiomeric substrates or products
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- 19 -
of the hydrvlase-catalyzed test reaction. Here, the test substrates can
be employed as a racemate. The separation may be effected both in
capillaries and by the use of prepared microchips which allow for
electrophoretical separation and parallel running of the analyses for a
high sample throughput. In both cases, it is a precondition that the
enantiomers can be separated by capillary electrophoresis.
The invention will now be further illustrated by the following Examples
and Figures.
Figure I shows the experimentally obtained measured curves for the
determination of the apparent enantioselectivity (Eapp) in the hydrolysis
of (R)- and (S~-2-methyldecanoic acid p-nitrophenyl ester with culture
supernatants of the lipase mutants P1B O1-E4, PZB 0$-H3, P3B 13-
D10, P4B 04-H3, P5B 14-C11, P4BSF 03~G10, and the wild type lipase
from P, aeruginosa (the slopes have the unit [mOD/min]).
Figure 2: Comparison of the DNA sequences of the lipase mutants P1$
O1-H1, P1B 01-E4, P2B 08-H3, P3B 13-D10, P4B 04-H3, P5B 14-C11 and
P4BSF 03-G10 Si55F with the sequence of the wild type of lipase from
P, aeruginosa (the mutated bases with respect to the wild type are
boxed, the origin of the mature lipase mutants is at base 163 or at
base 162 in the wild type).
In the following Example, the gene of the lipase from P. aeruginosa
(isolation according to K.-E. Jager, Ruhr-Universitat Bochum) has been
used for an optimization, the substrate for which the enantioselectivity
of the lipase was to be improved was (R,S)-2-methyldecanoic acid. A
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- as -
lipase mutant with a preference for the (S)-enantiorner was to be
developed. The screening test was performed with (R)- and (S)-2-
methyl-decanoic acrd p-nitrophenyl ester.
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- 21 -
Formula 5
(R,S)-2-methyldecanoic acid
Bacterial strains
E. cvli JMlp9:
e14-(McrA), re~Al, endAl, gyrA95, thi-_i, hsdRl7(rK-mK+),
supE44, relAi, o(lac-proAB), [F' trae36 proAB IacIq ZoM l S]
(Stratagene)
P. aeruginosa PABST7.1:
IacUVS/IacIq controlled T7-polymerise gene stably inte-
grated in the chromosome of strain P, aeruginosa PASS,
which bears a deletion in the structural gene of lipase IipA
(K.-E. Jaeger et al., J. Mol. Cat. Part 8, X997, in press)
Plasmid_s
pMutS: BamHI/Apal fragment (1045 bp) of the P. aeruginosa lipase
gene IipA in the vector pBluescript KSII (Stratagene)
pUCPL6A: BamHI/HindlIl fragment (2.8 kbp) comprising the P. aerugi-
nosa lipase operon in the vector pUCPKS (Watson et al.,
Gene 199fi, 172, 163) under the control of the T7 promoter
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22 -
Culturing of b~tgri~
E. eoli JM109 is grown over night (16 h) at 37 °C in 5 ml of LB
medium
an a test tube roller. For P, aeruginosa PABST7. i, 1 mM IPTG is added to
the medium. For the screening test, P. aeruginosa PABST7.1 is grown in
microtitration plates on a rotary shaker, the culture volume being
200 NI and the incubation being prolonged to 36-48 h. Antibiotics are
added in the following concentrations:
E coli JM109: ampicillin 100 pg/ml; P. aeruginosa PABST7.1: carbenicillin
200 pg/mi, tetracyclin 50 pg/ml
Mutagenizina PCR
The lipase gene IipA is amplified using the plasmid pMutS linearized
with endonuclease Xmn r as a template and the following PCR primers:
A: 5'-GCGCAATTAACCCTCACTAAAGGGAACAAA-3';
B: 5'-GCGTAATACGACTCACTATAGGGGGAA-~'
After purification of the PCR product using a Qiagen Qiaquick Column',
it serves as a template in a mutagenic PCR. The reaction conditions are
as follows: a 100 NI reaction volume contains 16.6 mM (Nhia)zSOa;
67 mM Tris-HCI {pH 8.8); 6.1 mM MgClz; 6.7 uM EDTA (pH 8.0); O.z mM
dNTPs; 10 rnM mercaptoethanol; 10 pl of DMSO; 10 pmol each of the
primers; 0.1 ng of template DNA; and 1 U of Taq polymerase {Goldstar,
Eurogentec). The reaction volume is covered with a layer of 100 pl of
paraffin. Ten parallel reactions were performed which were combined
after completion of the reaction. The cycling protocol is as follows: A
2 min denaturation at 98 °C is followed by Z5 cycles with 1 min at
94 °C, 2 min at 64 °C, 1 min at 72 °C on a Robocycler 40
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-- 2 3 -
(Stratagene), followed by incubation for 7 min at 72 °C. The Taq
polyrnerase is added after the denaturation of the 1st cycle. The
sequencing of the PCR products yields an error rate of about 1-2 base
substitutions per 1000 bp.
Cloning of the PC products
The PCR products are precipitated with ethanol and resuspended in
distilled water. After restriction with Apal and BamHI, the 1046 by
fragment formed is purified using a Qiagen Qiaquick Column° and
ligated into the correspondingly prepared vector pUCPL6A using T4
DNA ligase (MBI Fermentas) for 2 h at room temperature. The reaction
volume is diluted 1:5 and transformed into 200 NI of competent cells of
E, coli JM109 prepared by the method of Hanahan (J. Mol. Biol. 1983,
166, 557). For this purpose, the DNA and cells are stored on ice for 1 h
and incubated with shaking at 42 °C for 2 min and, after the addition
of 700 pl of LB medium, at 37 °C for 45 min. The cell suspension is
subsequently plated onto LB (ampicillin 100 Ng/ml) plates. Sixty
nanograms of the PCR product employed in the ligation reaction will
yield about 1500 colonies. All colonies are resuspended in sterile LB
medium, the plasmid DNA is purified and transformed into P. aeruginosa
PABST7.i by electroporation according to the method of Farinha and
Kropinski (FEMS Microbiol. Lett. 1990, 70, 221). The 96 wells of the
microtitration plates are inoculated with one colony each and treated
as described in Culturing of bacteria. To obtain the culture super-
natant, which is to be employed subsequently in the test for stereo-
selectivity, the microtitration plates are centrifuged at 4000 rpm for
30 min.
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- 24 -
Test for stereoselectivi~,y
The lipase-containic~g culture supernatants obtained by centrifugation
are pipetted in two aliquots into adjacent wells of a microtitration
plate. The test volume is 100 pl and is composed of the following
components (Table 2)
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- 25 -
T_",a__b_I_e Z
Composition of the reaction mixture in the test for improved enanti-
oselectivity of lipase mutants
(R) reaction I (S) reaction
50 NI of culture super- ~ 50 girl of culture supernatant
natant
40 NI of 1.0 mM Tris/HCI 40 NI of 10 mM Tris/HCI
buffer, pH 7.5 buffer, pH 7.5
NI of substrate solution 10 NI of substrate solution
[10 mg/ml (R)-2-methyl- [10 mg/ml (S)-2-methyl-
decanoic acid p~nitrophenyl decanoic acid p-nitrophenyl
ester in DMF] ~ ester in DMF]
After the addition of the Tris/HCI buffer to the supernatants, the
microtitration plate is incubated at 30 °C for about 5 min. After
addition
of the substrate solution, the reaction is continuously monitored for
IO min by spectrophotometry at 410 nm at 30 °C. From the linear rise
of the absorption curve, which is a measure of the constant initial rate
of the hydrolysis, the apparent enantioselectivity (Eapp) is determined.
Thus, the slopes measured in the linear region of the initial rates of the
reactions for the pair of enantiomers are divided by one another to
obtain the value of the apparent cnantioselectivity of the correspond-
ing lipase mutant.
Determination of stereoselectivitv by gas chromatograuhy
Selected positive clones are grown in 5 ml liquid cultures (LB medium),
and after centrifugation and removal of the bacterial pellet, the lipase-
containing supernatant is employed for the reaction. As the substrate,
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PAGE 2B
- 26 -
100 pl of a solution of racemic (R,S)-2-methyldecanoic acid p-
nitrophenyl ester (10 mg/ml in dimethylformamide) is used. This solu-
tion is admixed with 700 NI of 10 mM Tris/HCI buffer, pH 7,5. The
reaction is started by adding 100 pl of culture supernatant and per-
formed at 30 °C and 1000 rpm in Eppendort reaction vessels. After 2.5
h, samples of 200 ~I each are removed and transferred to an Eppen-
dorf vessel filled with 200 NI of dichloromethane. After the addition of
25 N! of 20% aqueous hydrochloric acid, the products and educts are
extracted (vortex shaker, 1 min). Finally, the organic phase is used for
gas-chromatographic analysis (GC). Separation of the enantiomers of
the free 2-methyldecanoic acid is achieved thereby.
Separation conditions of GC:
Instrument: Hewlett Packard 5890
Column: 25 m 2.6 DM 3 Pent ~-CD/80% SE 54
Detector: FID
Temperature: 230 °C inlet; 80-190 °C with 2 °C/min
Gas: 0.6 bar Hz
Sample quantity: 0.1 ml
Results (1st cycle
Of the about 1000 clones examined which had been obtained by
mutagenizing PGR from the starting DNA (wild type gene of lipase from
P. aeruginosa), 12 were identified to have an improved enantioselectiv-
ity over that of the corresponding wild type enzyme. Finally, 3 clones
were selected and their enantioselectivity determined by GC analysis.
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- 2? -
Ta lei
Selected lipase mutants with improved enantioselectivity (1st cycle)
M~~ usPP{S) VAPD(R) ~pP'I% ee E value 2}
[rnODtmin] (mODlmin] (by GG)I {calculated
conversion from GC)
Wild type21.8 14.9 1.5 2.4 I 15.3 1.1
P1 B 01-E4128.4 43.2 3.0 36.1 I 23.2 2.4
P1 B 01-F1278.8 35.7 2.2 14.1 I 30.5 9 .4
P1B01-H1 158.7 56.2 2.8 37.614.5 2.2
~aPP = R)
Vaav(S)IVava~
2) E =
In[1-c(1+eeP)]/In[1-c(1-eeP)]
with
c = conversion,
eeP =
ee value
of
the product
The DNA of the clone PiB pi-E4 served as the starting point for a new
cycle of PGR mutagenization. Thus, the plasmid pUCPL6A was isolated
from the clone and transformed into E. coli ~M109 as described above.
After the preparation of the plasmid DNA, the 1045 by fragment was
obtained by restriction with Apal and BamHI and subsequent purifica-
tion and ligated into the correspondingly prepared plasmid pMutS. After
transformation and plasmid isolation, this plasmid served as template
DNA in a mutagenizing PCR under the conditions as described above.
The DNA obtained from the mutagenizing PCR served to prepare a new
mutant library (2nd generation).
Re_s_ul_ts (2nd c cle
From the mutant library of the 2nd generation, about 2200 clones were
used for the screening test. Ten mutants with an improved enantiose-
lectivity over that of mutant PiB 01-E4 were identified. Two mutants
(P2B 04-G11 and P2B 08-H3) were examined more closely by GC
a nalysis.
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- 28 -
Table 44
Selected lipase mutants with improved enantioselectivity (2nd cycle)
Mutant V,Pp(S) V,Pp(R) F_,~')% ee E value Z)
[mODlmin][mODlmin~ (by GC)I (calculated
conversionfrom GC)
P28 04-G11224.9 52.3 4.3 47.8 l 3.4
30.0
P2B 08-H3 310.8 67.4 4.6 56.6 I 4.1
19.3
~) EaDP - VaPPO)NaPP(R)
2) E = In[1-c(1+eeP)]/In[1-c(1-eeP)] with c = conversion, eeP = ee value of
the
product -
Clone P2B 08-H3 was used for the next mutation cycle (3rd genera-
tion) .
Results (3rd cycle)
From the mutant library of the 3rd generation, about 2400 clones were
used for the screening test. One mutant (P3B 13-DiQ) with an im-
proved enantioselectivity over that of mutant P2B 08-H3 was identified.
It was examined further by GC analysis.
Selected lipase mutants with improved enantioselectivity (3rd cycle)
Mutant V,~(S) V,pp(R) E,~') % ee E value
2)
[mQDlmin][rnODlmin] (by GC)I (calculated
conversion from GC)
P3B 13-D10240.0 35.2 ~ 6.9 74.8 I 34.610.2
I ~ I
~aPD ~ VaPP~s)NaPP~R~
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- 29 -
2) E x In[1-c(1+eeP)J/Intl-c(1-eev)~ with c = conversion, eeP = ee value of
the
product
Results i(4th r~ycle)
From the mutant library of the 4th generation, about 2000 clones were
used for the screening test. Four mutants with an improved enanti-
oselectivity over that of mutant P3B 13-D10 were identified. They were
examined further by GC analysis.
Selected lipase mutants with improved enantioselectivity (4th cycle}
Mutant V,pP{S) Vapp{Rj E,~,')% ee E value
2)
[mODlmin][mQDlminj (by GC}I {calculated
conversion from GG)
P4B 04-H3355.6 26.5 13.4 81.0 I 20.011.2
P48 01-F2162.4 13.8 11.7 82.1 / 5.0 10.6
P4B 15-G1315.4 28.1 11.2 80.0 ! 18.010.7
P4B 15-H7288.0 25.1 11.5 78.4 I 22.010.2
1) ~aPP ~ VaPP~S)/VaaP~R)
2) E = In[1-C(1+eeP)J/In[1-c(1-eep)] with c ~ conversion, eeo = ee value of
the
product
The clone P4B04-N3 was inserted in the next mutation cycle (5~' generation).
Results ,5th cycle
From the mutant library of the 5th generation, about 5200 clones were
used for the screening test. Two mutants with an improved enanti-
oselectivity over that of mutant P4B 04-H3 were identified. They were
examined further by GC analysis.
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- 30 -
Table 7
Selected lipase mutants with improved enantioselectivity (5th cycle)
Mutant V,~pp(S) V,pP(R) E,~,')% ee E value
2)
[mODlmin][mODlmin] (by GC)I (calculated
conversion from GC)
P5B 14-C11 275.9 17.3 15.9 77.0143.0 13.7
P5B 08-F2 124.0 8.7 14.3 79.7 ! 40.3 15.1
~ I
1 J Capp = VaPpW~/ VaPDt K)
2) E = In[1-c(i+eeP))/In[1-c(i-eeP)] with c = conversion, eeP ~ ee value of
the
product
Senuencinq of the positive mutants
By sequencing the positive mutants, the mutations could be localized
within the lipase genes (see Figure 2), After assigning the base triplets
to the corresponding amino acids, the following amino acid substitu-
tions result with respect to the wild type lipase from P. aeruginosa:
PiB 01-H1: T103I (Thr103 ~ IIe103), S149G (Ser149 ~ GIy149}
P1B OI-E4: S149G (Ser149 -~ GIy149)
P2B 08-H3: S149G (Ser149 ~ G1y149), S155L (Ser155 ~ Leu155)
P3B 13-D10: S149G (Ser149 -~ GIy149), S155L (Ser155 -~ Leux55),
V47G (Va147 ~ GIy47)
P4B 04-H3: S149G {SCr149 ~ GIy149), S155L (Ser155 ~ LeulSS),
V47G (Va147 ~ GIy47), S33N (Ser33 ~ Asn33), F259L
(Phe259 ~ Leu259}
P5B 14-Cil: S149G (Ser149 ~ GIy149), 5155L (Ser155 3 Leu155),
V47G (Va147 ~ GIy47), S33N (Ser33 ~ Asn33), F259L
(Phe259 ~ Leu259), K110R (Lys110 -~ Arg 110)
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- 31 -
Mutants P1B O1-E4, P2B 08-H3 and P3B 13-D10 were deposited on July
16, 1997, with the DSMZ - Deutsche Sammlung von Mikroorganismen
and Zellkulturen GmbH, D~38124 Braunschweig, Mascheroder Weg ib,
under the designations of DSM i 1 658, DSM 11 659 and DSM 1 Z 659,
respectively.
Mutants P5B 14-C11 and P4B 04-H3 were deposited on July 20, 1998,
with the DSMZ - Deutsche Sammlung von Mikroorganismen and Zellkul~
turen GmbH, D-38124 Braunschweig, Mascheroder Weg ib, under the
designations of DSM i2 320 and DSM iz 3Z2, respectively.
. lh a 2
The protocols far the culturing of the bacteria, the mutagenizing PCR
and the test method for enantioselectivity are analogous to those of
Example 1. However, in this Example, the preparation of extensive
mutant libraries is effected by in vitro recombination.
The DNA used for the in vitro recombination is either generated by
mutagenizing PCR or obtained by combining the DNA from any number
of clones from one or more clone generations formed by repeated
mutageniiing PCR. If the PCR products of a mutagenizing PCR are the
starting point for obtaining DNA for the in vitro recombination, the
procedure is as follows: The PCR products of the mutagenizing PCR (see
Example 1) are purified, cleaved with the restriction endonucleases Apa
I and BamH I, ligated into the correspondingly cleaved vector pMUTS
and then transformed into E. coli JM 109. The plasmid DNA from ail
transformation clones is isolated. If some number of selected clones
from one or more generations of mutant clones are the starting point
for obtaining DNA for the in vitro recombination, then the plasmid DNA
of the vector pMUT5 is isolated and combined with the respective
variants of the lipase gene of P, aeruginosa. In both cases, the further
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- 32 -
procedure is as follows: Restriction with the endonuclease Pvu II yields
a 1430 by fragment which comprises the binding sites of primers A and
B already used in the mutagenizing PCR, in addition to the structural
gene for the lipase from P. aeruginosa. This fragment is purified and
cleaved into randomly generated fragments by incubation with deoxy-
ribonuclease I (DNase I from bovine pancreas). The size of the frag~
ments and the error rate of the subsequent reassembling can be
influenced by selecting the incubation conditions.
DNase I treatment
In a total volume of 100 NI, 3 Ng of Pvu II fragments in 50 mM Tris/HCI,
pH 7.5, i0 mM MgCl2 or 10 rnM MnClz, respectively, and 50 Ng/ml BSA is
incubated at 23 °C with 0.075 U DNase I for 10-25 min or 1-10 min,
respectively. The reaction is terminated by incubation at 93 °C for
min. Depending on the reaction time, fragments of smaller than
500 by to smaller than 10 by are obtained. In the Case where only a
particular range of sizes is used, these fragments can be obtained from
agarose gels by selective electro-blotting on DEAE membrane (accord-
ing to F.M. Ausubel et al., eds., Current Protocols in Molecular Biology,
John Wiley and Sons, 1989). After purification of the fragments by the
Qiagen Nucleotide Removal Kit~ (Qiagen), the following reassembling
reaction is performed,
Reassembling reaction
10-30 ng of the fragments derived from the DNase I restriction a re
subjected to the following PCR cycles in 75 mM Tris/HCI, pH 9.0, 20 mM
(NH4)ZSOa, 0.0i°/v (w/v) Tween~ 20, 1.5 mM MgClz, 0.2 mM dNTPs with
2 U Goldstar Taq polymerase (Eurogentec) in a total volume of 50 pl:
2 min at 94 °C, 40 cycles of 1 min at 94 °C, 2 min at 52
°C and 1 min
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- 33 -
at 7Z °C, finally 7 min at 7Z °C. The Taq polymerise is added
after the
1 minute denaturing step of the 1st cycle.
i Nl from the reassembling reaction is employed in a subsequent PCR
reaction, which is composed as described for the reassembling reaction
with the following differences: instead of the DNase I generated
fragments, 1 NI of the reassembling reaction is employed as the
template DNA. In addition, primers A and B in a concentration of 0.2 mM
and 10% dimethylsulfoxide are added. The cycling protocol is a s
follows:
2 min at 98 °C, 30 cycles of 1 min at 94°C, 2 min at fi4
°C, 1 min at
72 °C and finally 7 min at 72 °C; parallel runs are performed.
The PCR
products formed in these reactions are purified, restricted with the
Restriction endonucleases Apa x and Bam Hi and cloned as described in
the paragraph Mutagenizing PCR of Example ~..
Results ~(in vitro recombination~w
Twelve clones of the 1st generation of the mutant library obtained by
mutagenizing PCR (see Example 1) were used for the in vitro recombina-
tion. The following clones which had shown improved enantioselectivity
in the screening test were used:
P1B 01-AZ, P1B O1-A6, P1B 01-D2, P1B 01-D5, P1B 01-E1, P1B 01-E4,
P1B Q1-F3, P1B O1-F11, PIB 01-H1, P1B 01-H3, P1B 01-F12.
The DNA of these clones recombined according to the procedure
described above is cloned as stated in the paragraph Mutagenizing
PCR, and the culture supernatants are employed in the test for enanti-
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- 34
oselectivity. About 1000 recombinant clones were tested. The two
identified recombinants S2A OI-EL1 and S2A 02-G3 exhibit a significant
improvement of enantioselectivity over the best mutant of the 1st
generation (PiB 01-E4) from Example 1.
Tabl~8,
Selected lipase mutants with improved enantioselectivity (in vitro
recombination)
Mutant V,pp(S) V,pp(R) E,~') ~ ee E value
2)
[mODVmin]~mODlminJ (by GC)I (calc.f~om
conversion GC)
S2A 01-E11145.6 41.6 3.5 41.0127.0 2.8
S2A 02-G3 210.8 62.0 3.4 38.0 ! 23.02.5
J APP = VaPGWJI~apP~Kl
2) E = In[i-c(1+ee~))/In[i.-c(i-eeP}) with c = conversion, eeP = ee value of
the
product
x m le,~
Side-directed saturation mutagenesis in the amino acid position 155 of
lipase mutant P3B 13-D10:
Plasmids:
pMut5 13D10: BamHI/ApaI fragment (1046 bp) of the gene of
mutant P3B 13D10 for the lipase from P. aeruginosa in
pBluescript KS II
pMut50AK 13D10: Deletion of the AfIIII/KpnI fragment in pMut5 13D10
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- 35 -
A fragment of the gene for the lipase from mutant P3B 13D10 is ampli-
fied using plasmid pMut5 13D10, linearized by endonuclease XmnI, and
the following PCR primers:
A; S'-GCGGAATTAACCCTCACTAAAGGGAACAAA-3'
M: 5'-GGTACGGAGAATNNNCTGGGCTCGC-3'
where N represents A or C or G or T.
The reaction conditions are as follows: A 50 NI reaction volume contains
75 mM Tris/HCI, pH 9.0 (at Z5 °C); 20 mM {NHa)~S04; 1.5 mM MgCl2;
0.01% (w/v) Tween~ 20; 10% (v/v) DMSO; 10 pmol Of each of the
primers; 0.1 ng of the template DNA; and 2 U of Taq polymerase
(Goldstar, Eurogentec). The cycling protocol is as follows: A Z min
denaturation at 98 °C is followed by 30 cycles with 1 min at 94
°C,
2 min at b4 °C, 1 min at 72 °C on a Robocycter 40 (Stratagene),
followed by incubation for 7 min at 72 °C. The Taq polymerase is
added after the denaturation of the 1st cycle. After purification of the
PCR products by agarose gel electrophoresis and elution of the DNA
from the agarose gel using the Nucleospin Extract Kit (Macherey &
Nagel), it was used as a primer (socalled megaprimer) in a subsequent
PCR. Thus, the lipase gene is amplified on the plasmid pMut5aAK
13D10, linearized by endonuclease XmnI, using the following PCR
primers and the above described reaction conditions:
A: 5'-GGGCAATTAACCCTCACTAAAGGGAACAAA-3'
B (megaprimer): 5'-GCGTAATACGACTCACTATAGGGCGAA-3'
The reaction conditions and the cycling protocol are as described
above, except that 1-10 ng of the megaprimer is added to the reaction
mixture. The cloning of the PCR products is effected as described under
Cloning of the PCR Products.
CA 02298069 2000-O1-21
_. .._. i;i
FEB-24-00 14:05 FROM: ID: PfIGE 36
- 36 -
I r i n mu n n r ion P 1 0
From the mutant library of the saturation mutagenesis (3rd generation,
P3B 13-D10}, about 900 clones were used for the screening test. One
mutant (P4BSF 03-G10) with an improved enantioselectivity over that
of mutant P38 13-D10 was identified. Tt was examined further by GC
analysis.
Ta le 9
Selected lipase mutant with improved enantioselectivity (3rd genera-
tion, P3B 13~D10}
Mutant VaPO(S) Vpp(R) Ep~') % ee (by E value z)
GC)I
(mODlmin][mODlnrinj % conversion(calculated
from
GC)
P4BSF 384.7 25.3 15.2 87.3119.0 20.4
03-G10
taPP - vaPP~S~IVapD~R~
2) a = in[1-c(1+eeP)]/In[1-c(1-eec)] with c = conversion, eeo ~ ee vane of the
product
CA 02298069 2000-O1-21
FEB-24-00 14:06 FROM: ID: PAGE 39
- 37 -
Seauencing of the positive mut~n~,sL
By sequencing the positive mutants, the mutations could be localized
within the lipase gene (see Figure 2). After assigning the base triplets
to the corresponding amino acids, the following amino acid substitution
resulted with respect to mutant P3B 13-DiO:
P4BSF 03-G10 : L155F (Leu155 ~ Phe155)
Mutant P4BSF 03-G10 was deposited on ~uiy 20, 1998, with the DSMZ -
Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, D-
38i24 Braunschweig, Mascheroder Weg 1b, under the designation of
DSM 12 321.
CA 02298069 2000-O1-21
i;i
FEB-24-00 14:06 FROM: ID: PAGE 40
- 38 --
SEQUENCE LISTING
(1) GENERAL INFORMATION:
{i) APPLICANT:
(A) NAME: Studiengesellschaft TCohle mbH
(B) STREET: Kaiser-Wilhelm-Platz 1
(C) CIfY: Muelhei.m an dex Ruhr
{E} COUNTRY: Germany
(F) QOSTA1~ CODE (ZIP): 45470
(1i) TITLE OF INVENTION: A Process for the Prcperation and
Identification of Novel Hxdrolases Having Improved
Properties
(iii) NUMBER OF SEQUENCES: 71
{iv) COMPUTER RFADAF3LF, FORM:
(A) MEDIUM TYPE: Floppy disk
(8) COMPUTER: JAM PC compat~.bJ,e
(C) OPERATING SYSTEM: PG-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release X1.0, Version 11.30 (Ef0)
f2l INFORMATION FOR SEQ ID N0: 1:
{i) SEQUENCE CH11RACTERI5TIC5:
(A) ~.ENGxH: 30 bass paixs
(6) TYPE: nucleic acid
(C} STRANDEDNESS: unknown
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /dpsc = "synthetic DNA"
(xil SEQUENCE DESCRIPTION: SEQ ID N0: J.:
GCGCAATTRA CCCTCACTpA ACGGAACAAA 30
(2} INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARP,CTFRISTICS:
(A) LENGTH: 27 base pairs
(8) TYPE: nucleic acid
(C} STRANDEDNESS: unknown
(U) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "synthetic DNA"
(x:i.) SFQUF,NCF nESCR>;PTION: SEQ ID N0: 2:
GCGTAATi4CG AC'fCACTATA GGGCGAA 27
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE Cf~ARACTERISTICS:
(A) LENGTH: 1049 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
CA 02298069 2000-O1-21
ili
FEB-24-00 14:06 FROM: ID: PAGE 41
39 -
(ix) ~'E.'9TUf~E:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017
(ix) FEATURE:
(A) NAME/KEX: mat~eptide
(8) LOCATION:163..1017
(X1) SEQUENCE DESCRIPTION: SEQ ZD N0: 3:
GGATCCCCCG GTTCTCCCGG GCAGGACGCG CCCCTCGGCC
60
AAGGATTCGG
GCGATGGC'I'G
CCA TCAACCT GAGATGAGAA ATGAAG AAGAAGTAT CTGCTCCCC C1'C 111
CAAC
MetLys LysLysTyr Leul~euPro i~eu
-26-25 -20
GGC CTGGCC ATCGGTCTC GCCTCTCTC GCTGCCAGC CCTCTGATC CAG 159
Gly LeuAla IleGlyLeu AlaSexLcu AlaAlaSer ProLeuIle Gln
-15 -10 -5
GCC AGCACC TACACCCAG ACCAAATAC CCCATCGTG CTGGCCCAC GGC 207
Ala SeiThr TyrThrG1~ ThrLysTyx ProIleval LeuAlaHis Gly
10 15
ATG c'rcGGC TTCGACAAC ATCCTCGGG GTCGACTAC TGGTTCGGC ATT 255
Met LeuGly PheAspAsn IleLeuGly ValAspTyr.TrpPhcGly Ile
20 25 30
CCC AGCGCC TTGCGCCGT GACGGTGCC CAGGTCT11CGTCACCGAA G't'C 303
PrA SerAla LeuArgArg AspGl,yA1a GlnValTyr ValThrG,l,uVal.
35 40 45
AGC CAGTTG GACACCTCG GAAGTCCGC GGCGAGCAG 'rTGCTGCAA CAG 351
,
Ser GlnLeu AspThrSer GluValArg GlyGluGln .T_,eT.PG1 G1
n n
50 55 60
GTG GAGG111iATCGTCGCC CTCAGCGGC CAGCCCAAG GTCAACC:TGATC 399
Val GJ.uGl.uIleValAJ.aLEUSerGly GlnProI,y~ValllsnLeu Ile
65 70 75
CCC C11C11GCCE1CGGCGGG CC:~ACCATC CGCTACG1'CGCCGCCGTA CGT 447
G HisSer HisGJ.yG1y prcThrIle Ary~ryrVal AlaAlaVal Arg
l
y
~ 85 yf.~ 95
O
CCC GAGC;'I'GA'1'CGC1'TCC GCCATCAGC GTCGGCGCC CCGCACAAC CGT 4
95
Pro A.,pLcu IlclllaSer AlaI1eSer vaiGlyAla ProHisLys G1y
100 105 110
TCG GACACC GCCGACTTC CTGCGCCAG ATCCCACCC cGTTcGGcc GGC 513
Ser AspThr Ala.A.cpphP T,pnArgGln IleProPro GlySerAld Gly
115 120 125
GAG GCAGTC CTCTCCGCG CTGGTCAAC AGCCTCGGC GcGcTGATC:AGL 591
G.l.uAlaVal Lo_uSerGly LeuVa.lAs SNrT,a~iGl.yAlaLeuIle Ser
r~
130 ~3s i~o
TTC CTT TGC AGC GGr, c;c;c: Arc: GGT ACG CAG AAT TCA CTG GGC TCG CTG 639
Phc Lcu Scr Ser Gly Vly ~d~hr Gly Thr G1n Asn Sex Leu Gly Ser Leu
145 1~0 155
GAG 'i~CG CTG AAC Aec GAG GGT GCC GCG CGC 'rTC AAC GCC AAG TAC CCG 687
Glu Scr Leu Asn Ser Glu Gly A.la Ala Arg Phe Asn Ala Lys Tyr Pro
160 165 170 175
CA 02298069 2000-O1-21
FEB-24-00 14:06 FROM: ID: PAGE 42
- 40 -
CAGGGC A'1'CCCCACC TCGGCC TGCGGCGAA GGCGCCTAC GTC AAC 735
AAG
Gl.nGly IleProThr SerAla CysGlyGlu GlyAl,aTyr LysVal Asn
180 185 190
GGCGTG AGCTATTAC TCCTGG AGCGGTTCC TCGCCGCTG ACCAAC T'1'C7$3
GlyVal SerTyrTyr SerTrp SerGlySer SerProLeu ThrAsn Phe
195 200 205
C'TCGAT CCGAGCGAC GCCTTC CTCGGCGCC TCGTCGCTG ACCTTC AAG 831
i,euAsp ProSerAsp AlaPhe LeuGlyAl.aSerScrLeu ThrPhe Lys
210 21S 220
AACGGC AccGCCAAC GACGGC CTGGTCGGC AccTGCAGT TCGCAC CTG 879
AsnGly 'PhrAJ.aAsn AspGly LeuValGly ThrCysSer 5orIsisLeu
225 230 235
GGCATC GTGATCcGC GACAAC TACCGGATG AnccACCTG GACGAG GTG 927
GlyMet ValIleAxg AspAsn TyrArqMet AsnHisLeu AspGlu val
240 295 250 255
AACCAG GTCTTCGGC CTCRCC AGCCTGTTC GAGACCAGC CCGGTC AGC 975
AsnG;l.nvatPheGly LeuI'hrSerLeuPhe GluThrSer ProVal Ser
260 265 ?7Q
GTCTAC CGCCAGCAC GCCAAC CGCCTGRAG AACGCCAGC C'!'G 101.7
ValTyr ArgGlriHi.sAlaAsn ArgLeuLys AsriAlaSer Leu
275 780 285
TAGGACCCCG GCCGGGGCC1' CGGCCCGGGC CC 1049
(2) INFORMATION FICR SEQ J:D N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
t8) TYPE: amino acid
(D) TOPOLOGY: Zinear
(ii) MOLECULE TYPE: protein
(xi) SEQUk~NCE DESCRJPTION: SEQ ID NO: 4:
Mpt Lys Lys Lys Tyr Leu Leu Pro Leu Gly Leu Ala Ile Gly Leu Ala
-26 -25 -20 -I5
Ser Leu Ala AJ.a Ser Pro Leu Ile Glri Ala Ser Thr. Tyr Thr Gln Thr
-10 -5 1 5
Lys Tyr Pro Ile V~t i~Pls Ala His GIy Met Leu Gly Phe Asp Asn Ile
Z5 20
Leu Gly Vat Asp Tyr Trp Fhe Gly Ile Fro Scx l~la Leu Arg Arg A.sp
30 35
Gly Ala Gln VaJ. Tyr val Thr Glu Val Ser, Gln Leu Asp Thr Ser Glu
40 95 50
Val llrg Gly Glu Gln Leu Leu Gln Gln Val Glu G7,u Ile Val Ala Leu
55 60 65 70
Ser Gly Gln Pxo Lys Val Asn Leu Ile Gly Ftis Sex. His Gly Gly Pro
75 80 85
Thr. Il.e Arg Tyr val Ala iSla Val Arg Pz'o Asp Leu Ile Ala Ser Ala
90 95 100
CA 02298069 2000-O1-21
FEB-24-0O 14:06 FROM: ID: PAGE 43
- 41 -
ile Ser Val Gly Ala Pro His Lys Gly Ser Asp Th,r. Ala Asp Phe Leu
105 110 115
Arg Gln lle Pro Pro Gly Scr Ala Gly Glu Ala Va.7. Leu Ser ~Gly Leu
120 125 130
Val Asn Ser Leu Gly Ala Leu Ile Ser Phe Lcu Ser Ser Gly Gly '!'hr
135 140 145 150
Gly Thr Gln Asn Sex Lcu Gly Ser Leu Glu Ser Leu Asn Scr Glu Gly
155 7.60 165
Ala Ala Arg Phe Asn Ala Lys Tyr Faro Gln GJ.y .T.3.e Pro Thr Ser Ala
170 1W 180
Cys Gly Giu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr Tyr Ser Trp
185 190 195
Ser G,ly Ser Ser Pro Leu Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe
200 205 210
Leu G:l,y Ala ser Ser Leu '!'hr Phe Lys Asn Gly Thr Ala Asn Asp Gly
215 220 X75 230
Leu Val Gly Thr Cys Ser Sc:r His Leu Gly Met VaJ. I1e Arg Asp Asn
235 240 2~5
Tyr Arg Met Asn His Leu Asp Glu Va,~, Asn Gln Val phe Gly Leu Thr
250 255 260
Ser heu Phc Glu Thr Ser Pro Val Sex val Tyr Arg Gln His Ala Asn
265 270 275
Arg Leu Lys Asn Ala Ser Leu
280 285
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) Lk:NGTH: 1049 base pairs
(B) TYPE: nucleic acid
(C) STRANUEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DN11 (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(H) LOCATION:85..1017
( a. x ) FEATURE
(A) NAME/KEY: znat-peptide
(Bl LOCATION:163..101~
(xi) SEQUk:NCE DESCRIPTION: SEQ ID N0: 5:
GGATCCCCCG GTTCTCCCGG 1~GGA1~TCGG GGCATGGCTG GCAGGACGCG CCCCTCGGCC 60
CCATCAACCT GAGATGAGAA CAAC ATG AAG ~1AG AAG TC'J;' CTG CTC CCC CTC 111
Met Lys Lys Lys Sir Leu Leu Yro Leu
-76 -25 -20
GGC CT6 GCC ATC GGT CTC GCC TCT CTC GCT GCC AGC CCT CTG ATC CAG 159
Gly Leu Ala Tl.e Gly Lcu 111a Ser Leu Ala Ala Ser Pro Leu Ile Gln
-15 -10 ~5
CA 02298069 2000-O1-21
FEB-24-00 14:07 FROM: ID: PAGE 44
- 42 -
GCCAGC ACCTACACC CAGACCAAA CCC ATCCTGCTG GCCCACGGC 207
'1'AC
AJ,aSer ThrTyrThr Gln'rhrLys TyrPro IleValT.euAlaHisGly
1 5 1.0 15
ATGCTC GGCTTCGAC A11C1~.TCC'1'CGGGGTC GACTACTGG TTCGGCA'TT 7_55
MetLeu G1yPheAsp AsnI1eLeu Glyval AspTyr~I'rpPheGlyIle
20 25 30
CCCAGC GCCTTGCGC CGTGACGGT GCCCAG GTCT'ACCTC ACCGAAGTC 303
Proser AlaLeuArg ArgAspGly AlaGJ.nVa.I.Tyrval ThrGluVal
35 40 95
AGCCAG TTGGACACC TCGGAAGTC CGCGGC GAGcAGTTG CTGCAACAC; 351
SerGln LeuAspThr Ser,GluVa1 ArgGly GluGlnLeu LeuGlnGln
50 55 60
GTGGAG GAAATCGTC GCCCTCAGC GGCCAG CCCAAGGTC AACCTGATC 399
ValGJ,uGluIleVal AlaLeuSex GlyGln proLysVal AsnLeuIle
65 70 75
GGCCAC AGCCACGGC GGGCCGACC ATCCGC TACGTCGCC GCCGTACGT 447
GlyHis SerHisGly G.l.yProThr lleArg TyrvalAla AlaVaiArg
80 85 90 95
CCCGAC CTGATCGCT TCCGCCACC AGCGTC GGCGCCCCG CACAAGGCT 49S
P.ro,AspLeuIleAla SerAlaThr SerVal GlyAlaPro HisLysGly
100 105 110
TCGGAC nCCGCCGAC TTCCTGCGC CAGATC CCACGGGGT TCGGCCGGC 593
SerAsp ThrAlaAsp PheLeuArg GlnIle ProProGly SexAlaGly
115 120 125
GAGGCA GTCCTCTCC GGGCTGGTC 11110AGC CTCGGCGCG CTGATCAGC 591.
GluAla ValLeuSer GlyLeuVal AsnSer LcuGlyAla LeuzleSFr
130 135 1.40
1'TCCTT TCCAGCGGC GGCACCGGT ACGCAG AAT'1'CACTG CGCTCGCTG 639
PheLeu ser5erGly Gl,yThrGly ThrGln AsnSerLEU GlySerLeu
145 150 155
GAG'PCGCTGAACACC GAGGGTGCC GCGCGC TTCAACGCC AAGTAGCCG 687
GluSer LeuAsnSer GluGlyAla Alal~lrgPheAsnAla LysTyrPro
160 165 1~i0 1'75
CAG GGC ATC CCC ACC TCG GCC TGC GGC GAA GGC GCC TAC AAG GTC AAC 735
Gln Gl.y IJ_e Pro Thr Ser Ala Cys Gly Glu Gly Ala Tyr Lys val Asn
180 185 190
GGC GTG AGC TAT TAC TCC TGG AGC GGT TCC TCG CCG CTG ACC 11110 TTC 783
Gly Val Sex Tyr Tyr Ser Trp Ser Gly Ser Ser Pro Taeu Thr Asn Phe
195 200 205
CTC GAT CCG AGC GAC GCC TTC CTC GGC GCC TCG TCG CTG ACC TTC AAG 831
Leu Asp Pro Ser Asp Ala Phe Leu Gly Ala Ser Ser ,heu Thr Phe Lys
210 2I5 220
AAC GGC AcC GCC AAC GAC GGC cTG GTC GGC ACC TGC AGT TCG CAC cTG 879
Asn Gly Thr Ala Asn Asp GJ_y hpu Val Gly Thr Cys Ser Ser His Leu
22S 230 235
GGC ATG GTG A'i'C CGC GAC N1C TAC CGG ATG AAC C~1C CTG GAC GAG GTG 927
Gly Met Val Ilc llrg Asp Asn Tyr Arg Met A.sn His L~ru As,p Glu Val
240 245 250 255
CA 02298069 2000-O1-21
FEB-24-00 14:07 FROM: ID: PAGE 45
- 43 -
AAC CAG GTC TTC GGC CTC ACC AGC CTG TTC GAG ACC AGC CCG GTC AGC 975
Asn Gln Val Phe Gly Leu Thr Ser Leu Phe Gl,u Thr Ser Pro Val Scr
260 265 270
GTC TAC CGC CAG CAC GCC AAC CGC CTG AAG AAC GCC AGC CTG 1017
Val Tyx' Arg Gln His Ala Asn Arg Leu Lys Asn Ala Ser Leu
275 280 X80
TAGGACCCCG GCCGGGGCCT CGGCCCGGGC CC 1049
(2) INFORMATrON ~'OR SEQ ID N0: 6:
(i) SEQUENCE CHARACTF,RISTICS:
(AI LENGTH: 31.1 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TXPE: protein
(ri) SEQUENCE DESCRIPTrON: SEQ ID N0: 6:
Met hys J.,ys Lys Ser Leu Leu Pro Leu Gly Leu Ala IJ.e Gly Lcu Ala
-26 -25 -20 -15
Ser Leu Ala Ala Ser P,ro J,eu Ile Gln Ala Ser Thr Tyr Thr Gln Thr
-J.0 -5 1 5
Lys Tyr Pro Ile Va1 Leu Ala His Gly Met Leu Gly Phe Asp Asn I1~
15 20
Leu Gly vaJ. Asp Tyr Trp Phe Gly Ile Pro Sex AJ.a J.eu Arg Arg Asp
25 30 35
Gly Ala Gln val Tyr val Thr Glu val Ser G1n l,eu Asp Thr Scr G1u
40 A5 50
VaJ. Arg Gly Glu Gln Leu Leu Gln Gln val Glu Glu Ile Val Ala Leu
55 60 65 ?0
5er Gly Gln Pro Lys val Asn Leu Ile Gly His Ser Ha.s Gly G1y Pro
75 80 85
Th.r. Ile Arg Tyr val Ala Ala Val Arg Pxo Asp Leu Ile Ala Ser Ala
90 95 100
Thr Ser val Gly Ala Pro His Lys GJ.y Ser Asp Thr Ala Asp Phe Leu
105 110 115
Arg Gln IlE Pro Pro Gly Ser AJ.a Gly Glu Ala val Leu Ser Gly Lev,
120 7.25 130
Val Asn Ser Leu Gly Ala T~eu Ile Ser Phe Leu Ser Ser Gl~y GJ.y Thr
135 140 145 150
Gly Thr Gln Asn SEr Lcu Gly Ser Leu Glu Ser Leu Asn 5er Glu Gly
155 160 7.65
Ala Ala Arg Phe Asn Ala Lys Tyx Fro Cln Gly Ile Pro 'i'hr Ser Ala
1~0 175 180
C;ys Gly Glu Gly Al.a Tyr Lys val Asn Gly val Ser Tyr Tyr. SQL T~~
l90 ~~s
Ser Gly Ser Sez Pzo Leu Thr Asn Fhe Leu Asia Fro Ser Asp Ala Phe
200 205 210
CA 02298069 2000-O1-21
FEB-24-00 14:07 FROM: ID: PACE 46
- 44 -
Leu Gly Ala Scar Ser Leu Thr Phe Lys Asn Gly 'rhr Ala Asn Asp Gly
21S ?.20 225 230
Leu Val Gly Thr Cys Ser Ser H.is Leu Gly Met Val I.le Arg Asp Asn
235 240 245
Tyr Arg Met Asri His Leu Asp Gl.u Val Asn Gln Val Phe Gly Leu Thr
250 755 .7, 60
Sex Leu Phe Glu Thr Sex Pro Val Ser Val Tyr Arg G1n His Al.a Asn
265 270 275
Arg I,eu Lys Asn Ala Ser Leu
280 285
(2) INFORMA'T'ION FOR SEQ ID N0: 7:
(~.) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1099 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(i.i) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
{A) NAME/KEY: CDS
(B) LOCATION:85..1OZ~
(ix) FEATURE:
(A) NAME/KEY: mat~Ept:i.de
{H) LOCAT10N:163..1017
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
GGATCCCCCG GTTCTCCCGG AAGGA'iTCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 60
CCATCAACCT 11.7.
GAGA'1GAGAA
CAAC
ATG
AAG
AAG
AAG
T'C'1'
CTG
CTC
CCC
CTC
Met
Lys
Lys
Lys
Se,r.
Leu
Leu
Pro
Leu
~26 -20
-25
GGCcTGGCC ATCGGT CTCGcCTCT CTCGC'CGCC AGCCCTCTG ATCCAG 159
G1yLeuAla IleGly LeuAla5er l~euAlaAla SerProLeu IleGln
-15 -10 -5
GCCAGCACC TACACC CAGACCA~1ATACCCCATC GTGCTGGCC CACGGC 20'~
AlaSexThr TyrThr GlnThrLys TyrProIle V3.).LeuAla H.isGly
1 S 10 15
ATGCTCGGC TTCGAC AACATCCTT GGGGTCGAC TACTGGTTC GGCAT'1'255
MetLeuGly PheAsp AsnIleLeu GlyVallispTyr"1'rpPhe GlyIle
70 25 30
CCCAGCGCC TTGCGC CGTGACGGT GCCCAGGTC TACCTCACC GAAGTC 303
ProSerAla LeuArg Ar.gAspGly AlaGlnVal TyrValThr GluVal
35 40 45
AGCcAGT'rGGACACC TCGGAAGTC CGCGGCGAG CAGTTGCTG calACAG 351
SerGlnLeu AspThr ScrGluVal ArgGtyGlu GlnLeuLeu GlnGln
50 55 60
GTGGAGGAA ATCGTC GCCCTCAGG GGCCAGCCC AAGGTCAAC GTGATC 399
ValGluGlu IleVal Alaz~euSer GlyGlnPro LysvalAsn LeuIle
65 70 75
CA 02298069 2000-O1-21
FE8-24-00 14:0A FROM: ID: PAGE 47
45 -
GGCC.ACAGC CACGGCGGG CCGACC CGCTAC GTCGCCGCC GTA 497
A'fC CGT
GlyHisSer HisGlyGly ProThr Il,eArgTyr ValAlaAla val.Arg
80 85 90 95
CCCGACCTG ATCGCTTCC GCCACC AGCG'T'CGGC GCCCCGCAC AAGGGT 495
ProAspLeu IleAlaSer AlaThr ServalGly AlaProH,i.sLysGly
100 10.5 lI0
TCGGACACC GCCGACTTC CTGCGC CAGATCCCA CGGGGTTCG GCCGGC 543
SerlispThr AlaAspPhe r..euArg Gl,nIlcPro ProGlySer AJ.,aGly
115 120 125
GAGGCAGTC CTCTCCGGG CTGGTC AACAGCCTC GGCGCGCTG ATCAGC 591
GluAlaVal heuSerG1y LeuVal,AsnSerLeu GlyAlaLeu TleSer
1,30 135 140
TTCCTTTCC AGCGGCGGC ACCGGT ACGCAGAAT TTACTGGGC TCGCTG 639
PheLeuSer SerGlyGly ThrGly Thr.GlnAsn LeuLcuGly ScxLeu
145 150 155
GAGTCGCTG AACAGr:GAG GGTGCC GCGCGCTTC AACGCCnAG TACcCG 68~
GluSerLeu AsnSerGlu GlyAla AlaArgPhe AsnAlaLys TyI;Pro
160 7.65 170 175
CAGGGCATC CCCACCTCG GCCTGC GGCGAAGGC GCCT~1CAAG GTCAAC 735
G.l,nGlyIle ProThrSer nlaCys GlyGl,uGly AlaTyrLys ValAsn
180 185 190
GGCGTGAGC TATI'ACTCC TGGAGC GGTTCCTCG CCGCTGACC AACZ"1'C783
GlyValSex' TyrSer TrpSer GlySerSer ProLeuThr AsnPhe
z95 200 205
CTCGATCCG AGCGACGGC TTCcTC GGCGccTCGTcG CTGACC TTC 831
AAG
LeuAspPro SerAspAla PheLeu GlyAlaSerSer LeuThr PhcL.ys
21.0 215 220
AACGGCACC GCCAACGAC GGCCTG G'1'c:GGCACCTGC Ac:'rTCG CACCTG 879
AsnGiyThr AlaAsnAsp GJ,xLcu ValGIyThrCys SexSer HisLeu
2~~ 230 235
GGCATGGTG ATCCGCGAC AACTAC GGc;A'1'~AAGCAC CTGc:ACGAG~TG 9~7
GlyMetVat T1_AArgAsp AsnTyr ArgMctAsnIti9LrCLiAsp G1uvdl
240 295 ZSU 255
.~,ACGAGGTG rTCGGCCTC ACCAGC C'i'GTTCGAGACC AGCCCG GTCA~;C97
h
AsnGlnVal Phpc1_yLeu ThLSer LeuPheGIu'1'hrSPrFro ValSe.c
260 265 X70
GTCTnCCGC cAGc:ACGCC AACCGC CTGAAG~C GCC HGCCTC; I01?
valTyrArg GlnHisAla AsnArg L.euLysAsnAIa SerLeu
2~~ zao
TAGGACCCCG C:C 10q
GCCGGGGCCT y
CGGC;CC;UGGC
(%) INFORMATION FOR SEO Ip NO: 8:
(i) SEQUENCE CHARAC'1'~:RISTIf:S:
(A.) LENGTH: 311 amino acids
(B) TYPI~:: amino acid
(D) TOPOLOGY: linear
(ii) MOLECUhF. TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ZD N0. 8:
CA 02298069 2000-O1-21
ID: PAGE 49
FEB-24-00 14:09 FROM:
- 46 -
Met Lys Lys i,ys Ser Lcu Leu Pro Leu Gly Leu Ala I;Le Gly Leu Ala
--26 -25 -20 -15
Ser Leu ia.la A13 Ser Pro Lcu Ile Gln Ala Scr Thr Tyr Th.r Gln 'I~hr
-10 -5 1 5
Lys Tyr Pro Ile val Leu Ala His Gly Met Leu Gly Phe Asp Asn I.le
15 20
Leu Gly Val Asp Tyr Trp Phe Gly Ile Pro Ser Ala I~eu Arg Arg Asp
25 30 35
Gly Ala Gln Val Tyr Val Thr GIu Val Ser Gln Leu Asp Thr Ser Glu.
40 45 50
Val llrg Gly Glu Gln Leu Leu Gln Gln Val Glu Glu Ile Val Ala Leu
55 60 65 j0
Ser Gly Gln Pro Lys VaI Asn Leu Ile Gly Hi.s Ser His Gly G1y Pro
75 80 85
Thr Ile Arg Tyr Val AIa Ala Val Arg Pro hsp Leu Ile J~l,a Ser Ala
90 95 100
Thr Ser Val Gly Ala Pro His Lys Gly sex Asp Thr A.la Asp Phc Leu
I05 110 115
Axg Gln Ilc Pro Pxo Gly Ser Ala Gly Glu Ala Val Leu Ser Gly Leu
~. 20 125 130
Val Asn Ser Lcu Gly Ala Leu Ile Ser Phe Leu Ser Ser Gly Gly Thr
135 140 145 150
Gly Thr Gl_n Asn Leu Leu Gly Ser Leu G.lu Ser Leu Asn Sez' Glu Gly
7.55 160 165
Ala ALa ~lrg phe Asn Ala Lys Tyr Pro Gln Gly Ile Pro '!'hr Ser Ala
170 175 180
Cys Gly Glu Gly 111a Tyr Lyg Va.l, Asn Gly Val Ser Tyr Tyr Ser Trp
185 190 195
Ser Gly Ser 5er Pro Leu Thr Asn Phe Leu Asp L~ro Ser Asp Ala Phe
200 205 710
Leu Gly Ala Ser Ser Leu Thr Phe Lys Asn G7.y Thr Ala Asn Asp Gly
215 220 225 230
Lcu Val Gly Thr Cys Ser Ser. His Leu Cly Met Vai Il.e Arg Asp Asn
235 240 245
Tyr Arg Met Asn His Leu Asp Glu Val Asn G.7.,n Val J'he Gly Lcu Thr
250 255 260
Ser Leu Phe Glu Thr Ser Pr.o Val. Ser Val Tyr Arg Gln His Ala Asn
265 270 275
Arg heu Lys Asn Ala Sex Leu
280 285
(2) INFORMATION FOR SEQ ID NO: 9:
(i} SEQUENCE CHARACTERISTICS:
(A} LENGTH: 10!7 ba;;e pairs
CA 02298069 2000-O1-21
FEB-24-00 14:06 FROM: ID: P14GE 49
- 47 -
{B) TYPE: nucleic acid
{C) STRANDEDNESS: unknown
(D) TOPOJ,OGY: unknown
{ii) NJOLFCULE TYPE: vNA (genomic)
(iX) FEATURE:
{A) NAME/KEY: CDS
(R) LOCATION:84..1016
(.ix) FEATURE:
tA) NAME/KEY: mat~pept~.de
{8) LOCA'I'ION:162..1016
{xi) SEQUENCE DESCRINTION: SEQ ID N0: 9:
GGA'1'CCCCGG TTCTCCCGGA AGGATTCGGG CGATGGCTGG CAGGACGCGC CCCTCGGCCC 60
CATCAACCTG AGATGAGAAC AAC ATG AAG AAG AAG TCT CTG CTC
CCC CTC 110
Met Lys Lys Lys Ser Leu Lcu Pro Leu
-26 -25 -20
GGC CTG GCC ATC GGT CTC GCC TCT CTC GCT GGC AGC CCT CTG
ATC CAG 1.58
Gl,y Leu Ala Ile Gly Leu Ala Ser Leu Ala Ala Se
P
r
ro Leu Ile Gln
-15
-IO _g
GCC AGC .ACC TAC ACC GIG ACC AAA TAC CCC ATC GTG
CTG GCC CAC GGC 206
Ala Ser Thr Tyr Thr Gln Thx Lys '1'yr Pro I1
l
e Va
Leu Ala Flis G1y
1
'i
'
7. 5
ATG CTC GGC TTC GAC AAC ATC CTC GCG GTC GAC TAC TGG
TTC GGC ATT 25~
Met Leu Gly Phe Asp Asn Il.e Leu Gl
Val A
T
~
y
sp
yr, 1
rp Phe Gly Ile
30
CCC AGC GCC TTG CGC CGT GAC GGT GCC CAG GTC
1
'
T 302
~C G
TC ACC GAA GTC
Pro Ser Ala Leu Axg Arg Asp Gl
Ala GI
V
l
y
n
a
Tyr va,l Thr Glu Val
45
AGC CAG TTG GAC ACC TCG GAA GTC CGC GGC GAG CAG TTG
CTG CAA CAG 350
Ser Gln Leu Asp Thr Ser Glu Val Arg G~
y GIU Gl
,
n Leu Leu Gln Gln
60
GTG GAG GAA ATC GTC GCC CTC AGC GGC CAG CCC AAG GTC A
AC cTG ATC 398
Val Glu Glu IIe Val 111a Leu Ser Gl
Gl
p
y
n
ro I,ys val Asn Leu Tle
75
GGC C.~C AGC CAC GGC GGG CCG ACC ATC CGC TAC GTC GCC
GCC GTA CGT 446
G.ly His Ser His G7.y Gly Pro Thr Ile Arg T
r V
l Al
y
a
a Ala Val Arg
85
95
CCC CAC CTG ATC GCT TCC GCC ACC AGC GTC GGC GCC CCG CA
C AAG GGT 499
Pro Asp Leu Ile Ala Ser ala Thr Ser 'VaJ
Gl
Al
P
,
y
a
ro His Lys Gly
100
105 17.0
TCG cAC ncC GCC GAC TTC CTG CGC CAG IaTC CCA CCG
GGT TCG GCC GGC 542
Sex Asp Thr Ala Asp Phe Leu Axg Gln Il
P
e
ro Pro Gly Ser Ala Gly
115
120 125
GAG CCA GTC cTC Tcc GGG CTG GTC AAC AGC CTC Gf;r GC
G CTG ATC J~CC ;:90
GJ.u Ala Val Leu Ser Gly Leu Val A
S
an
ex Leu C;ly Ala Leu lle Se.r
130
1_i5 190
TTC cTT TCC AGC GGC AGC Acc GGT ACG cAG AAT Tr
A eT
~
_. 638
G GGC
rcG c:TG
Phe Lou Ser SNr Gly Ser ~rl,.r Cly Thr Gl
A
n
EI= J~_Y Leu Gly Ser Leu
195 15c)
155
CA 02298069 2000-O1-21
FEB-24-00 14:06 FROM: ID: PAGE 50
- 48 -
GAGTCGCTG AACAGCGAG GGTGCC GCGCGCT'TCAACGCCAAG TAGCCG 686
GluSerLeu AsnSerGiu GlyAla AlaArgPhe AsnAlaLys TyrPro
160 165 170
1
~5
GAGGGCATC CCCACCTCG GCCTGC GGCCAAGGC GCCT11CAAG G'fCAAC 734
G,lnGiyIle ProThrSer AlaCys GlyGluGly AlaTyrLys ValAsn
180 185 190
GGCGTGAGC TATTAGTCC TGG1~GCGGTTCCTCG CCGCTG11CCAACTTC 782
GlyValSer TyrTyrSer TrpSer GlySerSer ProLeu'ThrAsnPhe
195 200 205
CTCGATCCG ACCGAGGCC T'TCCTC GGCGCCTCG TCGCfGACC TxCAAG 830
LeuAspPro SerAspAla PheLeu GlyAlaSer SerLeuThr pheLys
z,.
o 215 220
AACGGCACC GCCAACGAG GGCCTG GTCGGCACC TGCAGTTCG CACCTG 878
AsnGl,yThr AlaAsnAsp G.lyLeu ValGlyThr CysSerSer HisLeu
225 230 235
GGCATGGTG ATCCGCGAG TAG CGGATGAAC CACCTGGAG GAGGTG 976
AAC
GiyMetVal IleArgAsp AsnTyr ArqMetF.snHisLeuAsp G1uVal
240 245 250
255
AACGAGGTC TTCGGCCTC ACCAGC CTGTTCGAG ACCAGCCCG GTCAGC 97~
AsnGlnVal PheClyLeu ThrSer LeuPheGlu ThrSerPro Val5er
260 265 270
GTCTAGCGC GAGCACGCC AACCGC CTGAAG1~4CGCCAGCCTG x026
ValTyrArg GinHisAla AsnArg LQULysAsn AlaSerLeu
27g 280 285
TAGGACCCCG C
GCCGGGGCC'f
CGGCCCGGGC
107
(2) INFORMATION fl~R Sk;Q ID NO: 10:
(i.) SEøUENCE CHARACTERISTICS:
(A} LENGTH : 311 am:i rrc, acids
(8) TYPE:; amino BCid
(U) ToPCLOGY: linaar
(ii) MOL~;CULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEø ID N0: 10:
Met Lys Ly3 Ly3 Sci Leu hev pro Leu Gly Leu At.tt Ile fly Leu Ala
-26 -2S --20 -15
Ser Leu Ala Ala Ser Pro Lcu Ile Glr~ Aid Spr Thr '.t'yr Thr Gln Thr
~la ~J ~ 5
l.ys Tyr Pro Ilo Val Leu A.le His Gi5 Met Leu Gly Fhe A3p Asn Ile
~n 2U
Leu Gly Val A3p Tyr Trp phe Gly Ile rro Ser Al:r Leu Arg Arg 1s.3,p
75 30 35
GJ.y Ala Gln Val Tyr val Thr Glu Val Ser Gln Leu Asp Thr Ser Glu
40 95 50
Val Arg Gly Glu Gln Leu heu Gin Gln Vai Glu Glu Ile Val Ala Leu
55 60 65 'I O
Ser Gly Gln Pro Lys Val, Asn Leu Il,e G1y His Ser H;~.s Giy Gly f'ro
CA 02298069 2000-O1-21
FEB-24-00 14:09 FROM: ID: PACE Sl
- 49 -
75 80 85
Thr Ile .Ard Tyr Val Ala A.la Val A95 Pro lap Leu Ile ~A.7.a Ser Ala
g0 100
Thr Ser Val Gl~r Ala pro His i~s0 Gly Scr Asp Thr A7,a Asp Phe Leu
105 115
Arg Gln Ile Pro Pro Gly Ser Al,a Gly Glu A.la Val Leu Ser Gly Leu
1.20
125 130
Val Asn Ser Leu Gly Ala Leu Ile Ser Phe heu Ser Ser. Gly Ser Thr
235 140 145 150
Gly Thr Gln Asn Ser Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly
i55 a60 7.65
Ala Ala Arg Phe Asn Ala Lys Tyr Pro Gln Gly Ile Pxo Thr Ser Ala
170 775 180
Cys Gly Glu Gly Ala ~'yr Lys vat Asn Gly Va.~. Ser Tyr Tyx Ser Trp
185 190 1.95
Ser Gly ser Ser Pro Lau Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe
200 20S 210
Leu Gly Ala Sex Ser Lcu Thr Phe Lys Asn Gly Thr AJ.a Asn Asp Gly
215 220 2z5 230
Leu Val Gly Thr Cys Scr Ser His Leu Gly Met val .rle Elrg Asp Asn
235 240 245
Tyr Axg Met Asn His Leu Asp Glu Val Aan Gln Val Phc Cly Leu Thr
X50
255 260
Ser Leu Phe Glu Thr Sex Fro val Ser Val Tyr Axg Gln His Ala Asn
265 270
Arg L~u Lys Asn Ala Se:r Leu
280 295
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERTSTrCS:
(A) LENGTH. 1049 base pai.xs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(,ii) MOLECULE TYPE: DNA (genomic)
(.ix) YEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017
( i.X ) FEATURE
(A) NAME/KEY: mat-peptide
(8) LOCATION:163..101'7
(xi.) S~;QUENCE DESCRTPTION: SFQ ID N0: 11:
GGATCCCCCG GTTCTCCCGG AAGGATTCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 60
CCATCAACCT GAGATGAG~ CAAC ATG AAG AAG AAG L'CT CTG CTC CCC CTC 111
CA 02298069 2000-O1-21
"~.,,.
FEB-24-00 14:09 FROM: ID: PAGE 52
- 50 -
Met Lys Lys Lys Ser L~u T,eu Pro L,eu
-26 -25 -20
GGC CTG GCCATC GGTCTCGCC TCTCTCGCT GCCAGCCCT CTGATCCAG 159
Gly Leu AlaIle GlyLeuAla SexLeuAlc~AlaSerP.roLeuIleGln
-15 -10 -5
GCC AGC ACCTAC ACCCACACC AlIATACCCC ATCG'PGCTG GCCCACGGC 207
Ala Ser ThrTyr ThrGlnThr LysTyrPro 11eVa1Leu AlaHisGly
1 5 10 15
ATG CTC GGCTTC GACAACATC CTTGGGGTC GACT11CTGG TTCGGCATT ?55
M2t Leu GlyPhe AspAsriIle LruG,lyval AspTyrTrp PheGIyI).e
20 25 30
CCC AGC GCCTTG CGCCGTGAC GGTGCCCAG GTCTACGTC ACCGAAGGC 303
Pra Ser AlaLeu ArgArgAsp G7.yAlaGln ValTyrval ThrGluGly
35 40 45
AGC CAG TTGGAC AGCTCGGAA GTCCGCGGC GAGCAGTTG CTGCAACAC 351
Sex Gln LeuAsp ThrSerGlu ValArgGly GluGlnLeu LeuG.lnGln
50 55 60
GTG GAG GAAATC GTCGCCCTC AGCGGCCAG CCCAAGGTC AACCTGATC 399
Val Glu GluIlc VaIAlaLeu SerGlyGln ProLysVal AsnLeuIle
65 70 75
GGC cAC AGCCAC GGCGGGCCG AcCATCCGc TACGTCGCC GcCGTACGT 447
Gly His 5orHis GlyGlyPro ThrIlcArg TyrvalAla AlavalA,rg
80 85 90 95
CCC GAC CTGATC GCTTCCGGC ACCAGCGTC GGCGCCCCG CACAAGGGT 495
Pro Asp Leu11e AlaSerAla ThrSc:rVal GlyAlaPro HisLysGly
100 105 110
TCG GAC ACCGCC GACTTCCTG CGCCAGATC CCACCGCGT TCGGCCGGC 543
Ser Asp ThrA1a AspPheLeu ArgGlnIle ProProGly SexAlaG1y
115 17.0 125
GAG GCA GTCCTC TCCGGGCTC GTCAACAGC CTCGGCGCG CTGATCAGC 591
Glu Ala ValLeu SerGlyLau ValAsnSer i,euGlyAla LeuIleSer
130 135 140
TTC CTT TCCAGC CCCCCCACC GGTACGCAG AATTTACTG GGCTc:GCrG 639
Phe Leu SerSex GlyGlyThr GlyThrGln AsnLeuLcu GlySerLeu
145 1:p0 155
GAG TCG CTGAAC AGCGAGGG'PGCCGCGCGC 'T"1CAACGCC AAGTACCCG 68'~
Glu Ser LeuAsn SerGluGly AlaAa.aArg PheAsnAia LysTyrPro
I60 165 170 175
cAG GGC ATCccc ACCTCGGcC TGCGGCGAA GGCGCCTAC AAGGTCAAC 735
Gln Gly IlePro ThrSerA,7,aCysGlyGlu GlyAlaTyr LysValAsn
3.80 185 190
GGC GTG AGCTAT TACTCCTGG AGCGG?TCC TCGCCGCTG ACCraACTTC 783
Gly vat SerTyr TyrSexTrp SerGlySex SerproLeu ThrAsnPhe
195 200 205
CTC GAT CCGAGC GACGCCTTC CTCGGCGCC 'I'CGTCGCTG ACC'rTCAAG 831
Leu Asp ProSer AspAlaPhe LeuGlyAla SerSerLeu ThrPheLys
210 215 220
AAC GGC ACCGCC AACGACGGC CTGGTCGGC ACCTGC11GTTCGCACCTG 879
Asn GJ.yThrRla AsnAspGly LeuvalGly ThrCysSer SerHisLeu
225 230 235
CA 9 00-O1-21
0229806 20
FEB-24-00 14:09 FROM: ID: PlIGE 53
- 51 -
GGC ATG GTG ATC CGC GAC lfAC TAC CGG ATG AAC CAC CTG GAC GAG GTG 927
Gly Met Val Ile Arg Asp Asn Tyr Arg Met Asn His Lcu Asp Giu Val
290 245 250 255
AAC cAG GTC TTC GGC CTC ACC AGC CTG TTC GAG ACC AGC cCG GTC AGC 975
Asn Gln Val Phe Gly Leu Thr Ser Leu Phe Glu Thx Ser Pro Val Ser
260 265 270
GTC TAC CGC CAG CAC GCC AAC CGC CTG AAG A1~C GCC AGC CTG 1027
Val Tyr Arg Gln His Ala Asn Arg Leu Lys Asn Ala Ser l,eu
275 280 28
TAGGACCCCG GCCGGGGCCT CGGCCCGGGC CC 1049
(2) INFORMATION EAR SE(~ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
($) TYPE: amino acSd
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: protein
(Xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Met Lys Lys Lys Ser Leu Leu Pro Leu Gly Leu Ala Ile Gly Leu Ala
-26 -25 -20 -15
Sex Leu Ala Ala Ser Pro L,eu Ile Gln Ala Ser Thr Tyr Thr Gln Th:t
~10 -5 1 5
Lys 'fyr Pro Ile Val Leu Ala His Gly Met Leu 61y Phe Asp Asn Ile
15 20
Leu Gly Val Asp Tyr Trp Phe G.l.y Ile Pro Ser Ala Leu Arg A,r.g Asp
25 30 35
Gly Ala Gln Val Tyr Val Thr Glu Gly Ser Gln Leu Asp Thr Scr Giu
90 45 50
Vai Arg Gly Glu GIn Leu Leu G,l.n Gln Val Clu Glu Ile Val Ala Leu
55 60 65 70
Ser Gly Gln Pro Lys Val Asn LQU Ile G80 His Ser His Gly Gl,y Pro
75 85
Thr Ile Arg Tyr Val Ala Ala Val Arg Pro Asp Leu Ile 111a Ser Ala
90 95 100
Thr Ser, val G1y Aia Pro His Lys Gly Ser Asp Th.r. AIa Asp Phe L,eu
105 110 115
Arg Gln Ile Pro Pro Gly Ser Ala Gly Glu Ala Val Leu Spr Gly Leu
770 125 130
Val. A3n Ser Leu Gly Ala Leu Iae Ser Phe Leu Ser Ser Gly Gly Thr
135 140 145 150
Gly Thr Gln Asn Leu Leu Gl,y Ser Leu Glu Se.r. Leu Asn Ser Glu Gly
1SS 160 165
Ala A1a Axg phe Asn Ala Lys Tyr Pro Gln Gly I1Q Pro Thr Ser Ala
170 175 180
CA 02298069 2000-O1-21
FEB-24-00 14:09 FROM= ID: PAGE 54
- 52 -
Cys Gly Glu Gly Ala Tyr Lys Vai Asn Gly Val Ser Tyr Tyr Set Trp
185 190 195
Ser Gly Ser Ser Pro Leu Thr Asn Phe Leu Asp Pro Ser Asp Ala Phe
200 205 21.0
Leu Gly Ala Scr Ser Leu Thr Phe Lys Asn Gly Thr 111a Asn Asp Gly
77.5 220 225 230
Leu Va,a_ Giy Thr Cys Ser Ser His Leu Gly Met Val Ile llrg Asp Asn
235 240 245
Tyr Arg Mgt Asn His heu Asp Glu Val Asn G7.n Val Phe Gly Leu Thr
250 255 250
Se.r. Leu Phc G~_u Thr Ser Pro Val Sex Val Tyr Arg Gln IIis Ala Asn
265 270 275
Arg Leu Lys Asn Ala Ssr Leu
280 285
(?) YNFORMATION FOR SEQ TD NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 1050 base pazrs
(H) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix} FEATURE:
(A} NAME/KEY: CDS
(B) LOCATION:85..1017
( 1X ) r'EATURE
(A) NAME/KEY: mat~eptide
($) LOCATION:163..L017
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 13:
GGATCCCCCG GTTCTCCCGG AAGGAT'TCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 6U
CCATCAACCT 111
GAGATGAGAlI
CAAC
ATG
AAG
AAG
AAG
TCT
CTG
CTC
CCC
CTC
Met
Lys
Lys
Lys
Ser
Leu
Leu
Pro
Leu
-26 -20
-75
GGCCTGGCC ATCGGTCTC GCcTcTCTC GCTGCCAGC CCTcTGATC CAG 159
GlyLeuAla IleGlyLcu AlaSerLcu AlaAlaSer ProLeuIlc GJ_n
-15 -10 -5
GCCAGC11CCTACACCCAG ACCAAATAC CCCATCGTG CTGGCCCAC GGC 207
AlaSe.r.1'hrTyrThrGln ThrLysTy1'ProIlcVa.l..T,euAlaHis Gly
1 5 10 15
ATGCTCCCC TI'CGACAAC ATCC'i'TGGG GTCGACTAC TGGTTCGGC ATT 255
MetLeuGly PheAspAsn IleLeuGly ValAspTyr TxpPheGly Ile
20 25 30
CCCAACGCC TTGCGCCGT GACGGTGCC CAGGTCTAC G'J'CACCGAA GGC 303
P.roAsnA:laLeuArgArg AspGl,yAla GlnVaJ.,'TyrValThrGJ.uGly
35 40 q5
AGCCAGTTG GACACCTCG GAAGTCCGC GGCGAGCAG TTGCTGCAA CAG 351
SerGlnLeu AspThrSe.rGluValArg GlyGluGln LeuLeuGln Gln
CA 02298069 2000-O1-21
FE8-24-00 14:10 FROM: ID: PACE 55
- 53 -
50 55 60
GTGG11GGAA ATCGTCGCC CTC1~GCGGCCAGCCCA1~GGTCAAC CTGATC 399
ValGluGlu IleValAla LeuSer GlyGlnProLys ValAsn L~uIle
65 70 75
GGCCACAGC CACGGCGGG CCGACC ATCCGCTACGTC GCCGCC cTACGT 447
GlyHisSer HisG,lyGly ProThr IleArgTyrVal AlaAl.aValArg
80 85 90 95
CCCGACCTG ATCGCTTCC GCCACC AGCGTCGGCGCC CCGCAC AAGGGT 495
Prohspheu IleAlaSer AlaThr 5erValGlyA1a l~rotiffsLysGly
100 lOS 110
TCGGACACC GCCGACTTC CTGCGC CAGATCCCACCG GGTTCG GCCGGC 543
SerAspThr AlaAspPhe LeuA.rgGlnIleProPro Gl.ySer AlaGly
17.5 120 a?,5
GAGGCnGTC CTCTCCGGG CTGGTC AACAGCCTCGGC GcGCTG ATCAGC a91
GluAlaVal LeuSerGly I,euVal AsnSerLeuGt,yA1aLcu IJ.eSer
130 135 140
TTCCTTTCC AGCGGCGGC ACCGGT ACGCAGAATTT11CTGGGC TCGCTG 639
PheLeuscr SerGlyGly ThrGly ThrGlnAsnLeu LeuGly SerLeu
145 150 155
GAGTCGCTG AACAGCGAG GGTCCC GCGCGCTTCAAC GCCAAG TACCCG 687
GluSerLeu AsnSerGlu GlyAla AlaArgPheAsn AlaLys '1'yrPro
lfi0 165 170 175
C11CCGCATC CCCACC'i'(:GGCCTGC GGCG11AGGCGCT TACI~AVG'1CAAC 735
GlnGlyIle ProThrSar AlaCys GlyGluGlyAla TyrLys ValAsn
180 185 190
GGCGTGAGC TATTACTCC TGGAGC GGTTCCTCGCCG CTGACC 1~ACTTC 783
GlyValSer TyrTyrSer TrpSer GlySerSerPro LeuThr AsnPhe
195 200 205
CTCGATCCG AGCGACGCC TTCCTC GGCGCCTCGTCG CTG11CCTTCAAG 831
LeuAspPro SerAspAla PheLeu GlyA.l.aSerSer Leu'1'hrPhcLys
2 10 215 220
AACGGCACC GCCAACGAC GGCc'rGGTCGGCnCCTGC AGTTcG CACCTG 879
AsnGlyThr AlaAsnlispGlyLeu Va1G.7,y'1'hrCys SerSer HisLeu
7?.5 230 235
GGCATGGTG ATCCGCGAC AACTAC CGGATGAACCAC CTGGAC GAGGTG 927
Gl.yMe~CVal IJ.eArgAsp AsnTyr ArgMetAsnHis LeuAsp GluVal
240 245 250 255
1~CCAGGTC CTCGGCC'1'CACCAGC C'TGTTCGAGACC AGCCCG GTCAGC 975
AsnGlnVal i.euGly~,euThrSer l~euPheGluThr SerPro Va1Ser
260 265 270
GTCTACcGC CAGCACGCC AACCGC CTGAAAAACGCC AGCCTG 1017
ValxyrArg GlnHisAla AsnArg LeuLysAsnAla SerLPu
275 280 285
'i'r~GGACCCCG CCG 10~U
GCCGGGGCCT
CGGCCCGGGC
(7 INFORMATION E'ORSEQ In 4:
) N0:
1
CA 02298069 2000-O1-21
FEB-24-00 14-10 FROM: ID: PAGE 56
- 54 -
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
(H) TYPE: am~,no acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(.ci) SE(~UENCE DESCRIPTION: SEQ ID N0: 14:
Met Lye Lys Lys Ser Leu Leu Pro Leu Gly Leu 111a Ile Gly Leu Ala
~26 -25 -20 -15
Ser Leu Ala Ala Ser Pro Leu Ile Gln Ala Ser Thr Tyr Thr Gln Thx
-10 -5 I 5
Lys Tyr Pro Ile Val Leu Ala His Gly Met Leu Gly Phe Asp Asn Ile
I5 20
Leu Gly Val Asp 'fyr Trp Phe Gly Ile Pro Asn 111a Leu Arg Arg Asp
~5 30 35
Gly Ala Gln Val Tyr Val Thr GJ.u Gly Sex. Gln Leu Asp Thr Ser Glu
40 95 50
Va,l Arg Gly Glu Gln Leu Leu G.l,n Gln Va7. Glu Glu Tle Val Ala Leu
55 60 65 70
Scr Gly Gln Pro Lys Val Asn Leu Ile Gly His Ser His Gly Gly Pro
75 80 ~ 85
Thr Ile Arg Tyr Val Ala Ala Val Arg Pro Rsp Leu Ile Al.a Ser Ala
90 95 100
Thr Ser val Gly A1a Pro His Lys Gly Ser Asp Thr Ala Asp Phe Leu
lOS lI0 115
Arg Gln Ile P.ro Pro Gly Ser Ala Gly Glu Al.a Val Leu Ser Gly Leu
120 125 130
Val Asn Ser Leu Gly Ala J~eu Ile Ser Phe Leu Sex Ser Gly Gly Thz~
135 190 I45 150
Gly Thr Gln Asn Leu Leu Gly Ser Leu Gl.u Ser Leu Asn Ser G.i.u Gly
155 160 165
Ala Ala Arg Phe Asn Ala L~ys Tyr Pro Gln Gly Ile Pro Thr. Ser l~la
170 175 180
Cys G.7_y Glu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr Tyr Ser Trp
1,85 190 195
Ser Gly Ser Ser Pro Lau 'fhr 7lsn Phe Leu lisp Pro Ser Asp Ala Phe
200 205 210
Leu Gly Ala Ser Ser Lau Thr hhe Lys Asn Gly Thr Ala Asn Asp Gly
215 220 225 230
Leu Va.l. Gly Thr Cys Ser Scr His Leu Gl.y Met Val Ile Arg Asp Asn
235 240 245
'1'yr Arg Met Asn His Leu Asp Glu Val Asn GJ.n Val Leu G7.y Leu Thr
2a0 255 260
Ser Leu Phe Glu Thr Ser Pro Val Ser Val Tyr Arg G1n His Al~a Asn
26S 270 275
CA 02298069 2000-O1-21
FEB-24-00 14:10 FROM= ID= PAGE 57
- 55 -
llz;g Leu Lys Asn Ala Ser Leu
280 285
(2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1099 base pairs
(B) TYPE: nucleic aced
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATI:ON:85..1017
(ix) FEATURE:
(A) NAME/KEY: mat~epti,de
(B) LOCATION:163..1017
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: I5:
GGATCCCCCG GTTCTCCCGG AAGGATTCGG GCGATGGCTG GCAGGACGCG CCCCTCGGCC 60
CC11TCA,A.CCT GAGATCAGAA AAG TCTCTG CTCCCC CTC 111
CAAC 11AG
ATG AAG
Met Lys LysSe.r,heu LeuPro Leu
Lys
-z6-25 -20
GGCCTGGCC11TCGGTCTC GCCTCTCTC GCTGCCAGCCCT CTGATC CAG 1$9
GlyLeuAlaIle GlyLeu AlaSerLeu AlaAlaSerPro LeuIle Gln
'15 -10 -5
GCCAGCACCTAC AccCAG ACCAAATAC CCCATCGTGCTG GcccAC GGC 207
l~laSerThrTyr ThrGln ThrLys'TyrProIIevalLcu AlaHis Gly
1 5 10 15
AT'GcTCGGCTTC GACAAC ATCCTTGGG GTCGACTACTGG TTCGGC ATT z55
MetLeuGlyPhe AspAsn IIeLeuGly valAspTyrTrp PheGly Ile
20 25 30
CCCAGCGGCTTG CGCCGT GACGGTGCC CAGGTCTACGTC ACCGAA GGC 303
ProSerAlaLeu ArgArg AspGlyAla GlnVal'1'yrvat ThrGlu Gly
35 90 45
AGCCAGTTGGAC ACCTCG GRAGTCCGC GGCGAGCAGTTG CTGCAA CAG 351
SerGlnLeuAsp ThrSpx GluvalArg G.tyGluGlnI,euLeuGln Gln
50 55 60
GTCGAGGAAATC GTCGCC CTCAGCGGC CAGCCCAAGGTC AACC~'GATC 399
valGluGluzle Val.Ala LeuScarG,lyGlnProLysVal l~snLeu Tle
65 70 75
GGC CAC AGC CAG GGC GGG CCG ACC ATC CGC TAC GTC GCC GCC GTA CG'T 497
Gly His Ser His Gly Gly Pro Thr Ilc Arg Tyr val A13 Ala Val Arg
80 85 90 95
CCC GAC CTG ATC GCT TCC GCC ACC AGC GTC GGC GCC CCG CAC AGG GGT g95
Pro Asp I,eu Ile A1a Ser. Ala Thr Ser vat Gly Aln Pxo His Arg Gly
100 105 110
TCG GAC ACC GCC GAC TTC CTG CGC CAG ATC GCA CCG GGT TCG GCC GGC 593
Ser Asp Thr Ala Asp Phc heu Arg Gln Ile Pro Pro Gly Ser Ala G1y
CA 02298069 2000-O1-21
FEB-24-00 14:10 FROM: ID: PAGE 5A
56 _
115 120 125
GAGGCA GTCCTCTCC GGGCTG G'tCAACAGC CTCGGCGCG CTCATC AGC 591
GluAla ValLPUSer GlyLeu ValAsnSer LeuGlyAla LeuI1e 5cr
130 135 140
TTCCTT TCCAGCGGC GGCACC GGTACGCAG AATTTACTG GGCTCG CTG 639
PheLeu SCrSerGly GlyThr.GlyThxGln AsnLeu>'.,euGlySer Leu
195 150 155
GAGTCG CTGAACAGT GAGGGT GCCGCGCGC TTCAACGCC AAGTAC C:CG68~
GluSex'LeuAsnSer GluGly AlaAlaArg PheAsnAla I,ysTyr Pro
160 165 170 17S
CAGGGC ATCCCCACC TCGGCC TGCGGCGAA GGCGCTT.~CAAGcTC A~aC735
GlnGly IlcProThr SerAla CysGlyGJ.uGlyAlaTyr LysVal Asn
180 185 190
GGCGTG AGCTATTAC TCCTGG AGCGGTTCC TCGCCGCTG ACCAAC TTC -~83
GlyVal SerT'yrTyr SerTrp SerGJ.ySer SerProLeu 't'hrAsn Phe
7.95 200 205
CTCGAT CCGAGCGAC GCCTTC CTCGGCGCC TCGTCGCTG ACCTTC AAG 831
LeuAsp ProSerAsp AlaPhe L~uGlyAla SerSerJ.,euThrPhe Lys
77.0 215 220
AACGGC ACCGCCAnC C11CGGC CTGGTCGGC ACCTGCAGT TCGC:aCCTG 879
AsnGly ThrAlaAsn AspGly LeuValGly ThrCysScr SerHis Leu
225 230 235
GGCATG G'1'GATCCGC GACAAC TACCGGATG AACCACCTG GACGAC G2'G927
G.lyMet VallleArg AspAsn TyrArgMet AsnIlisLeu AspGlu Val
240 245 250 755
AACCAG GTCCTCGGC CTCACC AGCCTGTTC GAGACCAGC CCGGTC AGC 975
AsnGln ValLeuGJ.yLeuThr SerLcuPhe GluThrSer ProVal Ser
260 265 27p
GTCTAC CGCCAGCAC GCCAAC CGCCTGAAG .SACGCCACC CTG 1017
ValTyr ArgGl.nHis AlaAsn ArgheuLys AsnAlaSex Leu
275 280 285
TAGGACCC CG GCCCGGGCCC 1049
GCCGGGGCCT
CG
(2) INFORMATION FOR SEQ ID NO: 16:
(.i? S~;QUENCE CHARACTFRISTZCS:
(A) LENGTH: 311 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(i.i.) MOLECULE 'TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 16:
Met Lys hys Lys 5el~ Leu Leu Pro Leu Gly i,eu Ala Ile Gly Leu Ala
-26 -75 -?.0 -7.5
Ser Leu Alx Ala Ser Pro Leu Zle Gln Ald Ser Thr Tyr Thr Gln 'Phr
-10 -g 1 5
Lys Tyr Pro IJ.e Val Leu Ald His G25 Met Lcu Gly Phe A..~,p Asn I:le
20
Leu Gly Vdl Asp Tyr Trp Phe Gly 71e Pro Ser A7a r,~" Arg Arg Asp
25 30 35
CA 02298069 2000-O1-21
i
ID: PAGE 59
FEB-24-00 14:11 FROM:
..
GJ.y Ala Gln val Tyr val Thr Glu Gly Sex Gln Leu Asp Thr Ser Glu
40 q5 50
Val Arg Gly Glu Gln Le:u Leu Gln Gln val Glu Glu Ile Va.l Ala Leu
55 60 65 70
Ser Gly Gln Pro Lys val Asn Leu Zle Gly His Ser His Gly Gly Pro
75 80 85
Thr Ile Arq Tyr Val Ala Ala val Arg Pro Asp Leu Ile Ala Sex Ala
90 95 I00
Thr Ser VaJ. Gly A1a Pro His Arg Gly Ser Asp Thr Ala Asp Phe Leu
105 110 1.15
Arg Gln Ile Pro Pro Gly Ser Ala Gly Gl.u Ala val Leu Ser Gly Leu
170 125 130
Val Asn Ser J~eu Gly Ala Leu Il.e Ser Phe I,eu Sor Ser Gly Gl,y Thr
135 140 145 150
Gly Thr Gln Asn Leu Lcu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly
1S5 160 165
Ala Ala Arg Phe Asn Ala Lys Tyr Pro Gln Gly Ile Pro Thr Ser Ala
1.75 7.80
Cys Gly Glu Gly Ala Tyr Lys Val Asn Gly Val Ser Tyr 'fyr Se.r Trp
185 190 195
Ser Gly Ser Ser Pro Leu Thr Asn Phe Lcu Asp Pro Sex Asp 111a Phe
200 205 210
Leu Gly Ala Ser Ser I,eu Thr Phc hys Asn Gly Thr Ala Asn Asp Gly
215 220 225 230
Leu Val Gly Thr Cys Ser Ser His Leu Gly Met Val Tle Arg Asp Asn
245
235
Tyr Arg Met Asn His l,eu Asp Glu Val Asn Gln Val Leu Gly Leu Thr
250 255 260
Ser Leu Phe Glu Thr Ser Pro Val Sex Val Tyr Arg Gln His Al.a Asn
265 2.'70 2 ~5
Arg Leu Lys Asn ALa Ser Leu
280 785
(2) INFORMATION FOR SEQ ID N0: 17:
(i) SFQU~;NCE CHARAC1'I;ItISTICS:
(A) J.ENGTH: 1049 base pairs
(H) TYPk'.: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
( i.i ) MOLECULE TYPE : DNA ( genom ic; )
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:85..1017
(ix) FEATURE:
(A) N11ME/KEY: mat peptide
CA 02298069 2000-O1-21
FEB-24-00 14:11 FROM= ID: PACE 60
58 ~-
(B) LOCATION:163..1017
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7.7:
GGA~i'CCCCCG GrTCTCCCGG CCCCTCGGCC
60
AAGGATTCGG
GCGATGGCTG
GcAGGACGCG
CCATCAACCT GAGATGAGRA 11TGAAGAAG AAGTCTCTG CTCCCCCTC 111
CAAC MetLysLys LysSerLau LeuProLeu
-26-25 ~20
GGCCTG ATC CTCGCC 'rCTCTC GCCAGC CTG CAG 159
GlyGCC GGT LeuAla SerGCT AlaccT ATC Gt7.n
Leu Ile -10Lcu Ser Leu
Ala Gly Ala Pro Ile
-15 -5
GCCAGC ACCTACACC cACACC AAATACCcc ATCGTGcTG GCCcACGGC 207
Al,aSer ThrT'yrThr GlnThr LysTyrPro IloValLeu A.l,aHisGay
1 5 J.
0 15
J1TGCTC GGCTTCG11CAACATC CTTGGGGTC GACT71CTGG TTCGGCATT 255
MetLeu GlyPheAsp Asnza.eLeuGlyVal lispTyrTrp PheGlyLle
20 75 30
CCCAGC GCCTTGCGC cGTGAC GGTGCCCAG GTCTACGTC ACCGAAcCC 303
ProSer Al,aLeuArg ArgAsp GlyAlaGln ValTyrVa,~ThrGluGly
35 40 ~!5
AGCCAG TTGGACACC TCGGAA GTC:CGCGGC GAGCAGTTG CTGCAl1CAG 351
SerGln LeuAspThr SerG7.uValArgCly GluGlnLeu LcuGJ.nGln
50 55 60
GTGGAG GAAATCGTC GCCCTC AGCGGCCAG CCCAAGGTC AACCTGATC 399
Va,1_Glu GluIleVal AlaLeu SerG.tyGln ProJaysVal Asn,LeuI1e
65 70 75
GGCCAC AGCCACGGC GGGCCG ACCATCGC TACGTCGCC GCCGTACGT 4~7
GlyHis Se'rHisGly GlyC ThrIleArg TyrValAla AlaValArg
80 85 Pro 90 95
CCCGAC C'i'G11TCGCT TCC AGCGTC.GGCGCCCCG CACAAGGG'r 4
ProAsp J,euIleAla GCG Serval GlyAlaPro H:isLysGly 95
100 ACC 105 110
Ser
Ala
Thr
TCGGAC nCCGCCGAC TTCGTGC;GCCAGATC.CCA CCGGGT TCGGCC:GGC
593
SerAsp ThT~AlaAsp PheLeuArg GlnIlePro P,-oGly SerAlaGl
y
115 120 125
GAGGCA GTCCTCTCC GGGCTGGTC AACAGCCTC GGCGCG CTGATCAGC 5
91
GluAla ValT~euSex GlyLeuVa,~,AsnSerLeu GlyAla LeuIleSer
130 135 140
TTCCTT 'i~CC11GCGGC GGCATCGGT ACGC11GAAT TTTCTG GGCTCGCTG
639
PheLeu SerSerGly GlyIleGly ThrGlnAsn PheLeu GlySerLeu
145 150 155
GAGTCG CTGAACAGC GAGGGTGCC GCGCGCTTC AACGCC AAGTACCCG 687
GluSer LeuAsnSer GluG:LyAla AlaArgPhe AsnAa.aLysT P
1 r r
o
60 165 170 y .
.
175
CAGGGC ATCCCCACC TCGGCCTGC GGCGAAGGC GCCTAC AAGGTCAAC 735
Gl
n Gly IleProThr SerAlaCys GlyGluG~.yAlaTyr LysVall\sn
180 185 190
GGCc;TGAGrTATTAC TCCTGGAGC GG'1'TCCTcc c:CGCTr AccnACTTC
783
GlyVaI 5r:rTy.Tyr SerTxpaer GlySers~r PrnLeu ThrAsnPhe
CA 02298069 2000-O1-21
FEB-24-00 14:11 FROM: ID: PAGE 61
59 -
195 200 205
CTCGAT CCGAGCGAC GCCTTC C'1'CGGCGCC '1'CGTCGCTG ACC'fTCAAG 837
L
.
eu Asp Pro5erAsp AlaPhe LeuGlyAla SerScr.Leu ThrPheLys
21
0 215 2Z0
AACGGC ACCGCCAAC GACGGC CTGGTCGGC ACC'fGCAGT TCGCliCCTG $i9
A '
sn Gly 1 AlaAsn AspGly LeuValGly ThrCysSer SerH;isLe
hr
u
225 230 235
GGCATG GTGATCCGC GACAAC TACCGGATG AACCACCTG CACGAGGTC 977
24 Met ValZleArq AspAsn TyrArgMet AsnHisLcu AspGluVal
y
0 245 250 255
AACCAG GTCT'PCGGC CTCACC AGCcTGTTC GAGnCCAGC CCGGTCAGC 975
AsnGln ValPheGly LeuThr SerLeuPhe GluThrSer ProValSer
260 265 770
GTCTIaCCGCCAGCAC GCCAAC CGCCTGAAG 11ACGCCAGC CTG
x.017
Va,lTyr ArqGInHis AIailsnArgLeuLys AsnA1aScx'Leu
275 280 285
TAGGACCCCG CCGGGGCC T GGGCCC
G CGGCCC
1049
(2) INFORMlITION FOR SEQ ID N0: 1$:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOhECULE TYP);: protein
(xi) SEQUENCE DESCRIPTION: SFQ ID N0: 18:
Met Lys Lys Lys Sex Leu Leu Pro Leu Gly Leu Ala Ilc Gly Lpu Ala
-?6 ,25 -20 -15
Ser Leu Ala Ala Ser Pro Leu lle Gln Ala Ser Thr Tyr Th.r. Gln 7.'hr
-10 _5 1 5
Lys Tyr Pro I1P Val Leu Ala His Gi5 Met Leu Gly Phe Asp Asn Iie
20
Leu Gly Val, Asp Tyr Trp Phe Gly Ile Pxo Ser Ala Leu Arg Arg Asp
25 30 35
Gly Ala Gl,n Val Tyr Val Thx Glu C~,l.y Ser G7 n T~ru Asp Thr Ser Glu
40 ~5 50
Val Arg G1y Qlu Gln Leu Leu Cln Gln Vr~l Glu Glu Ile Val Ala l;,eu
55 b0 55 70
Ser Gl,y Gln Pro Lys Val Asn Leu Ile Gly Kic Cor Hi.s Gly Gly Pro
75 00 $5
'i'hr lle Arg Tyr Val Ala Ala Val llrg pro Asp Leu Ile Ala Ser Ald
90 55 100
Thr Scr val Gly Ala PL'U His LVS Gly Ser Asp Thr Al.s~ Asp Phe Leu
105 110 115
Arg C;ln Ile Pr.~ Prn Gly Sor Al,~ GIy CJ.u Ala Val LCU Ser Gly Leu
120 125 130
Val lien Ser Leu Gly Ala Leu Ile 58r the Leu Ser Spr c:l.y ,ly Zls
CA 02298069 2000-O1-21
FEH-24-00 14:11 FROM: ID: PACE 62
' 60
135 140 145 150
GlyThr GlnAsnPhe T,euGly SerLeuGlu SerLcuAsn SprGlu Gly
7.55 160 165
AlaAla ArgPhoAsn nlaLys TyrFaroG.lnGlyIlePro 'PhrSer Ala
170 175 180
CysGly C~7.uGlyAla Tyr,Lys ValAsnG7,yValSerTyr TyrSer Trp
185 190 195
SerGly SerSerPro LeuThr AsnPheLeu AspPxoSer AspAla Phe
200 205 210
LeuGly AlaSerSc~xLeuThx PhcLysAsn GlyThrAla AsnAsp Gl.y
715 2z0 225 230
LeuVa.lGlyThrCys SerSer HisLcuGly MctVdlIle ArgAsp Asn
235 290 245
TyrArg MetAsnHis LeuAsp GluValAsriGlnValPhe GlyLcu 'L'hr
250 255 260
SerLou PheGluThr SexPro ValSerVaJ.'TyrArgGln HisAla Asn
265 270 975
AxgLeu LysAsnAla SerLeu
280 285
(2) rNFORMATION FOR SE:O ID N0: 19:
(i} SEQUENCE CHARACTERISTICS:
(A) L~;NGTH: 30 base pairs
(g) TYPE: nucJ.eic acid
(C) STRANDEDNESS: unknown
(D} TOPC?LOGY: linEar
(i..i) MOLECULE TYPE: other nucleic acid
{A) DESCRIPTION: /dese = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SF.Q ID NO: 19:
GcGC:AATTAA ccCTCACTAA AGGGAncAAA 30
(2) INFORMATION FOR SEQ ID N0: 20:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 25 base paixs
{B) TYPE: nucleic acid
(C) STRAhDEDNESS: unknown
(D) TOPOLOGY: linear
(.ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTTON: /desc = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 20:
GGTACGCAGA 1~x'NNNCTGGG C TCGC
(2} INFORMA':CrON FOR SEQ ID N0: 21:
(i} SEQUENCE CHARACTERISTICS:
{A) LENGTH: 27 base pairs
CA 02298069 2000-O1-21
ii
b
FEB-24-00 14_12 FROM: ID: PAGE 63
-- 61 -
(B) TYPE: nucleic acid
(G) STRANDEDNESS: unknown
(b) TOPOLOGY: linear
(ii) MOLECUhE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "synthetic DNA"
(xi) SEQUENCE DESCRIPTION: SEQ ZD N0: 21:
GCGTAATACG ACTCACTA1'A GGGCGAA
CA 02298069 2000-O1-21
