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METHOD FOR STUDYING PROTEIN INTERACTIONS IN VIVO
10
BACKGROUND
. The study of interactions between proteins in living cells is often
necessary to
understand the proteins' functions and their mechanisms of action. These
interactions are
currently' stud~ie~i using° immuno-precipitation, the yeast two hybrid
method; and ~-gal
complementation method.
ZO However, these methods are associated with several disadvantages. For
example, these methods are associated with false positives. Second, they do
not permit the
determination of quantitative information regarding the interactions. Further,
they do not
allow for in vivo real time monitoring of the interactions.
Therefore, it would be advantageous to have another method of studying
interactions between proteins in vivo, which does not have these
disadvantages. Further
preferably, the method could be used with a wide variety of proteins and in_a
wide variety of
living cells. Also preferably, the method could be used to determine the
interactions between
molcxules other than proteins.
SUMMARY
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method for determining whether a first protein interacts with a second pmtein
within a living
cell. The method comprises providing the first protein complexed to a donor
luciferase and
the second protein complexed to an acceptor fluomphore within the cell. The
donor
luciferase is capable of luminescence resonance energy transfer to the
acceptor fluorophore
when the first protein is in proximity to the second protein. Then, the
complexed first
protein and the complexed second protein are allowed to come into proximity to
each other
within the cell. Next, any fluorescence from the acceptor fluorophore is
detected.
Fluorescence of the acceptor fluorophore resulting from luminescence resonance
energy
transfer from the donor iuciferase to acceptor fluorophore the indicates that
the first protein
has interacted with the second protein.
In a preferred embodiment, providing the first protein complexed to a donor
luciferase and the second protein complexed to an acceptor fluorophore
comprises genetically
engineering DNA and transferring the genetically engineered DNA to the living
cell causing
the cell to produce the first protein compiexed to a donor luciferase and the
second protein
complexed to an acceptor fluorophore. In a particularly preferred embodiment,
the cell
which is provided with the first protein complexed to a donor luciferase and
the cell which is
provided with the second protein complexed to an acceptor fluorophore are
mammalian cells.
In another preferred embodiment, the donor luciferase provided is Renilla
luciferase. In yet another preferred embodiment, the acceptor fluorophore
provided is an
Aequorea green fluorescent protein.
In a particularly preferred embodiment, the det~don of acceptor fluorophore
fluorescence is performed using spectrofluoronlettry.
DESCRIPTION
The present invention includes a method for determining whether a first
protein interacts with a second protein in a living cell using luminescent
resonance energy
transfer {L.RET). Luminescence resonance energy transfer results from the
transfer of
excited state energy from a donor luciferase to an acceptor fluorophore. In
order for LRET
to occur, there must be an overlap between the emission spectrum of the donor
luciferase and
the excitation spectrum of the acceptor fluomphore.
The efficiency of luminescence resonance energy transfer is dependent on the
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Generally, significant energy ttnnsfers occur only where the donor hicifcrase
and acceptor
fluorophore are less than about 80 A of each other. This short distance is
considerably less
than the distance between for optical resolution between two entities using
conventional microscopy. Therefore, detecting luminescence resonance energy
transfer
between a donor hiciferase and an acceptor fluorophore indicates that the
donor luciferase
and acceptor fluorophore have come within the distance noeded for LRET to
occur, that is
less than about 80 A of each other.
The present invention utilizes luminescence resonance energy transfer to
determine whether an interacEion takes place between a first protein and a
second protein in a
living cell. This is accomplished by complexing a first protein to the donor
luciferase and
complexing the second protein to the acceptor fluorophore and placing the
complexed fast
protein and the complexed second protein in the cell under conditions suitable
for an
interaction between We first protein and the second protein to take place. If
the first protein
interacts with the second protein, the donor luciferase will come close enough
to the acceptor
fluorophore for luminescence resona~e energy transfer to take place and the
acceptor
fluorophore will fluoresce. Detection of fluorescence from the acceptor
fluorophore will,
thereby, indicate that the first protein has interacted with the second
protein.
Advantageously, this method allows for the detection of interaction between
the first protein
nd the second protein even though the interaction cannot be detected by
optical methods
such as conventional microscopy.
There are several advantages of using luminescent resonance e~rgy transfer to
detect the interaction between two proteins accordiwg to the present
invention. First, the
specific labeling of the proteins in living cells can be achieved through
generic engineering
methods where the introduction of fluorescent dyes into living cells is very
difficult.
Further, fluorescent dyes photobleach quickly while light emission of a
luciferase such as
Renilla luciferase originates from an enzymatic reaction that is relatively
stable if substrate
and oxygen are supplemented.
As used in this disclosure, "complexing a first protein to the donor
luciferase"
refers to joining the donor luciferase to the first protein in a manner that
the donor luciferase
and the first protein stay in essentially the same proximity to one another
during interaction
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the acceptor fluorophore° refers to joining the acceptor fluorophore to
the second protein in a
manner that the acceptor fluorophore and the second protein stay in
essentially the same
proximity to one another during interaction between the first protein and the
second protein.
Such complexing can be done, for example, by genetically engineering the cell
to produce a
fusion protein containing the donor luciferase and first protein, and the
acceptor fluorophore
and the second protein.
In a preferred embodiment, the present invention uses Renilla luciferase as
the
donor luciferase and "humanized" Aequorea green fluorescent protein
('humanized' GFP) as
the acceptor fluorophore. Renilla luciferase is a 34 kDa enzyme purified from
Renilla
reniformis. The enzyme catalyzes the oxidative decarboxylation of
coelenterazine in the
presence of oxygen to produce blue light with an emission wavelength maximum
of 471 nm.
Renilla luciferase was used as the donor luciferase because it requires an
exogenous substrate
rather than exogenous light for excitation. This, advantageously, eliminates
background
noise from an exogenous light source and from autofluorescence, and allows
easy and
accurate quantitative determination of light production.
'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation
maximum at 480 nm. It has a single amino acid difference from wild-type
Aequorea green
fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluomphore
because its
~- ~ excitation spectrum overlaps with the emission spectra of Renilla
luciferase: Additionally;
emissions from 'humanized' GFP can be visualized in living cells. Further,
'humanized'
GFP is expressed well in the mammalian cells transfected with 'humanized' GFP
cDNA that
were used to demonstrate this method.
The method for determining whether a first protein interacts with a second
protein according to the present invention was demonstrated as follows. In
summary,
insulin-like growth factor binding protein 6 (IGFBP b) and insulin-like growth
factor II (IGR
II) were selected as the first protein and second protein. IGFBP 6 is a
protein known to havE
a marked binding affinity for IGF-II.
The Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized'
GFP cDNA was fused to IGF II cDNA. Living cells were transfected with the
fused cDNA
and the fusion proteins were expressed. Cell extracts were produced and mixed.
The
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was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused
'humanized' GFP-IGF-II protein was detected. Demonstration one method
according to the
present invention will now be described in greater detail.
A) The Cloning of Fused IGP'BP-6 cDNA to Rtnilla Lucifierase cDNA; Fused IGF
II
S cDNA to 'hmnanized' GFP cDNA; and Fused Insulin cDNA to 'humanized' GFP
cDNA:
First, three fused cDNAs were produced: 1) fused IGFBP-6 cDNA and Renilla
luciferase cDNA; 2) fused IGF-II cDNA and 'humanized' GFP cDNA; and 3) fused
insulin
cDNA and 'humanized' GFP cDNA. IGFBP-6 cDNA, SEQ ID NO:1, GenBank accession
number MG90S4, encoded IGFBP-6, SEQ ID N0:2, which was used as the first
protein.
Renilla luciferase cDNA, SEQ ID N0:3, GenBank accession number M63S01, encoded
Renilla luciferase, SEQ II3 N0:4, which was used as the donor luciferase. IGF-
II cDNA,
SEQ ID NO:S, encoded IGF-II, SEQ ID N0:6, which was used as the second
protein.
'Humanized' GFP cDNA, SEQ ID N0:7, GenBank accession numberUS0963, encoded
'humanized' GFP, SEQ ID N0:8,which was used as the acceptor fluorophore.
Insulin
1S cDNA, SEQ ID NO:9, accession number AH002$44, encoded insulin, SEQ ID
NO;10.
Insulin, fused to 'humanized' GFP, was used as a control protein because
insulin is
homologous to IGF-II, but it does not bind to IGFBP-6. The IGFBP-6 cDNA, SEQ
ID
NO:1, IGF-II cDNA, SEQ ID NO:S, and insulin cDNA, SEQ ID N0:9, were modified
using PCR as follows. --
First, the cDNA of prepro-IGF-1I carried on an EcoRI fragment was cloned
into pBluescript KS (+) II vector. The insert was sequenced using T7 and T3
primers and
confirmed to contain the known cDNA sequence of prepro-IGF-IT. The S' end of
the IGF-II
precursor was connected to the T7 promoter in the pBlueseript KS (+) II
vector. An IGF-II
3' primer was designed to ge~rate a Notice of Allowance restriction site, to
remove the D
2S and E domains of prepro-IGF-II, and to maintain the Notice of Allowance
fragment of the
'humanized' GFP in frame with the open reading frame of IGF-Q.
Next, the IGF-II fragment was amplif:ed with PCR using the T7 promoter
primer and the IGF-II 3' primer. The PCR-amplified IGF-II fragment was
digested by
EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA,
US)
producing pCDNA-IGF-II. Then, the Notice of Allowance fragment of the
'humanized'
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The eDNA for precursor of insulin, which contained a signal peptide the B, C
and A domains, was modified in a manner corresponding to the IGF-II fragment,
above.
The 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin
cDNA to
produce pC-INS-GFP.
Finally, IGFBP 6 cDNA was amplified by PCR from a plasmid named
Rat-tagged human IGFBP6. The stop codon of IGFBP 6 was removed and the open
reading
frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC
(Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of
recombin~urt
Renilla luciferase. in transgereic plants results in high levels of light
emission. The Plant
Journal 1995;7;1031-$). The linking of the Renilla luciferase cDNA to the 3'
end of
modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC.
The sequences of the insert DNA fragments from ail the constructs were
verified by DNA sequencing analysis. Qiagen Maxi Plasmid Kit (Qiagen, Inc.,
Valencia,
CA) was used for the purification of plasmid DNA.
B) Transient Transfection of Mammalian Cells With pC-IGF-II-GFP, pC-INS-GFP
and
pC-IGFBP 6-RUC Using the Calccium Phosphate Precipitation Method:
Next, msunmalian cells were transfected with the clod fusion DNAs. First,
COS-7 cells (African green monkey kidney cell, American Type Culture
Collection CRL
_ 1651) were grownat 37 C in Dulbecco's Modified Eagle Medium (DMEM) with
L-Glutamine supplemented with 1096 fetal bovine serum and antibiotic
antimycotic solution
containing a final concentration of penicillin 100 unit/ml, streptomycin 100
mglmi and
amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% C02.
Groups of
1x106 of these cells were plated the day before transfection and were
approximately 5096 to
60 k confluent at the time of transfection.
Forty mg of each plasmid fusion DNA were precipitated and resuspended into
Dulbecco's Phosphate Buffered Saline Solution and the plasmid fusion DNAs was
introduced
into mamnnalian cells using the standard calcium phosphate precipitation
method.
Transfection efficiency was estimated by fluorescence microscopy after 24
hours. The
number of green fluorescent cells per plate were comparable in plates of pC-
IGF-II-GFP
DNA transfected cells, pC-INS-GFP DNA transfeeted cells and cells transfected
with a
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C) Confirmation of Expression of ~~sion Proteins:
Twenty-four hours after DNA transfection using DNA calcium phosphate
precipitation method, individual plasmid DNA transfected COS-7 cells were
visualized using
fluorescence microscopy by detection of GFP fluorescence. pC-IGF-II-GFP and
pC-INS-GFP transfected cells showed similar fluorescence patterns typical of
secretory
protein translocated thmugh ER to Golgi. The pC-IGFBP 6-RUC transfected cells
did not
fluoresce. However, the pC-IGFBP 6-RUC transfected cells did show luminescence
using a
low light imaging system after the addition of coelenterazine.
Further, the presence of fusion proteins IGF-II-GFP.and IGFBP 6-RUC,
having the expected molecular weights of about 36 kDa and 56 kDa,
respectively, were
detected using immunoblot analysis. This confirmed the presence of both fusion
proteins in
the transiently transfected cells.
D) Detection of Protein Interactions by Spedrofluorometry:
Having confirmed the presence of the expected fusion proteins iGF-II-GFP
and IGFBP 6-RUC, and the function of the donor luciferase and acceptor
fluorophore, cell
extracts from these transiently transfected cells were used to carry out a
protein binding assay
based on energy transfer between the Renilla luciferase and 'humanized' GFP
moieties of the
fusion proteins. Forty-eight hours after calcium transfection, the COS cells
were washed
twice with PBS and harvested using a cell scraper in luciferase assay buffer
containing 0.5 M
NaCI, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5. The harvested cells
were
sonicated 3 times for 10 seconds with an interval of IO seconds using a Fisher
Model 550
Sonic Dismembrator (Fisher Scient~c, Pittsburgh, PA, US) to produce cell
extracts.
Next, the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed
and 0.1 pg of coelenterazinc was immediately added. Spectrofluorometry was
performed
using a SPEX FluoroMaxm (Institunents S.A., Inc., Edison, NJ). The spectrum
showed a
single emission peak at 471 nm, which corresponds to the known emission of
Renilla
luciferase.
Following the first spectrofluommetry, the mixtures were kept at room
temperature for 30 minutes and the spectra wore traced again after fresh
coelenterazine was
added. The trace at 30 minutes showed two peaks with emission maxiimum at 471
nm and
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spectral pattern did not change over time.
Control cell extract mixtures from cells transfected with pC-INS-GFP and
pC-IGFBP 6-RUC were made similarly and their spectra traced. The traces showed
only one
peak at 471 nm, which corresponds to the emission peak of Renilla luciferase.
The spectral
pattern did not change over time.
Therefore, these data demonstrated that IGFBP 6 and IGF-II interacted but
that insulin and IGFBP 6 did not interact.
In addition to the above disclosed examples, protein-protein interactions were
also detected by the detection of LRET using corresponding methods in E. coli
cells and
mammalian cells which were co-transformed.
Although the present invention has been discussed in considerable detail with
reference to certain preferred embodiments, other embodiments are possible.
For example,
the interaction between molecules other than proteins could be studied by
corresponding
methods. Such other molecules could be provided to the living cell by
diffusion, infusion,
and incorporation or by other means. Further, fusion proteins produced from
genetically
engineered living cells could have post translational changes, such as the
addition of sugar
moieties, before their interactions are studied. Also; living~cells~can
be.visualized using
these methods by spectrofluorometry by low light image analysis in cells,
colonies and
tissues. Additionally, high thmugh put screening of colonies can be
accomplished using the
present methods combined with cell sorting and low light video analysis of
micro titre dishes
or multiple array detection. Therefore, the spirit and scope of the appended
claims should
not be limited to the description of preferred embodiments contained herein.
CA 02341314 2001-12-05
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CA 02341314 2001-12-05
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Dset Thr Ser Lys Val Tyr 7lap pro Glu Gln ~lrg Lys llrg list Ile Thr
1 5 10 15
Gly Pro Gla Trp Trp l~la 7lrg Cya Lys Gla ltet flan Val Leu llsp 8er
ZO Z5 30
Hhe Its Aaa Tyr Tyr 7lap 8er Glu Lys 8ia Ala Glu 7lsa Ala Val Ile
35 !0 !5
phe Lou 8is Gly llsn Ala ~11a 8er 8er Tyr Leu Trp llrg 8is Val Val
50 55 60
Pro 8is Ile Glu pro Val Ala 7lrg Cys Ile Ila pro Asp Leu Ile Gly
65 70 75 80
llet Gly Lya 8er Gly Lys Ssr Aly Asn Aly 8er Tyr llrg Leu Leu llsp
8S 90 95
81s Tyr Lys Tyr Lau Thr hla Trp phe Glu Leu Leu llsn Leu Pro Lys
100 105 110
Lys Ile Ile Phe Val Gly 8ia llsp Trp Gly 111a Gys Lsu Ala Phe 8ia
115 1Z0 ls5
Tyr Ser Tyr Glu 81s Gln hsp Lya Ile Lys Ala Ile Val 81s Ala Glu
130 135 1!0
Sar Val Val Aap Val Ila Alu 8er Trp flap Glu Trp pro Asp Ile Alu
CA 02341314 2001-12-05
wo oon4i~~ rcrius99noZO~
145 150 155 160
Alu Asp Ile Ala Leu Ile Lys Ser Alu Olu Aly A1u Lys list Val Leu
165 190 195
c~lu Asa Ara Phe Phs Val Olu Thr llst Lsu Pro 8sr Lys ils list Arg
180 185 190
Lys Leu Alu Pro Alu Glu Phe Ala Ala Tyr Leu Alu Pro phs Lys Alu
195 Z00 Z05
Lys Aly Alu Val Arg Arg pro Thr Leu Ser Trp Pro ArQ Alu ile Pro
Z10 115 Z~0
Leu Val Lys Oly Aly Lys Pro Asp Val Val Ala Ile Val Arg Asn Tyr
Z~5 Z90 i35 140
Asn Ala Tyr Lau Arg Ala Sar Asp Asp Lsu Pro Lys lsat Phe Ile Alu
Z45 Z50 Z55
Bar Asp Pro Aly Phs Phe Bar Asn Ala ile Val Alu Aly Ala Lys Irys
Z60 Z6s Z70
Phe Pro Asa Thr Alu Phe Val Lys Val Lys Aly Leu His Phe Ssr Aln
Z75 Z80 Z85
Alu Asp Ala Pro Asp Alu llet Aly Lys Tyr Ile Lys Ser Phe Val Alu
Z90 Z95 300
Arg Val Leu Lys Asa Gilu Aln
3Q5 310
<a10> 5
<Z11> 543
cZl2> DNA
<~13> Homo sapisas
<ZZO>
<aal> cDs
<222> il)..(543)
<400> 5
atg gga atc cca atg ggg aag tcg atg ctg gtg ctt ctc acc ttc ttg 48
liet Gly Tla Pro lief Aly Lys Bar ldst Lsu Val Leu Leu Thr Phe Leu
1 5 10 15
gcc ttc gcc tcg tgc tgc att get get tac cgc ccc agt gag acc ctg 96
CA 02341314 2001-12-05
wo oo~iar~i Pccrtrs~9noZO7
Ala Phe Ala 8er Cys Cys Zle Ala Ala Tyr Arg Pro 8er Glu Thr Lau
20 Z5 30
tgc ggc ggg gag ctg gtg gac acc ctc cag ttc gtc tgt ggg gac cgc 144
Cya lily Lily Glu Leu Yai Asp Thr Leu Oln phe Vai Cys Oly Asp Arg
35 40 45
ggc ttc tac ttc agc agg acc gca agc cgt gtg agc cgt cgc agc cgt 192
lily Phe Tyr Phe 8er Arg pro Ala 8er Arg Yal 8er Arg Arg 8er Arg
50 55 60
ggc atc gtt gag gag tgc tgt ttc cgc agc tgt gac ctg gcc ctc ctg a40
aly Ile Val Glu Glu Cys Cys phe Arg 8er Cyr Asp Leu Ala Leu Leu
65 70 75 80
gag acg tac tgt get acc cec gec aag tec gag agg gac gtg leg acc Z88
Cilu Thr Tyr Cys Ala Thr Pro Ala Lys 8er flu Arg Asp Val 8er Thr
85 90 95
cct ccg acc gtg ctt ccg gac aac ttc ecc aga tac ccc gtg ggc aag 336
Pro Pro Thr Val Leu pro Asp Asa Phe Pro Arg Tyr pro Val Qly Lys
lao ios 110
ttc ttc caa tat gac acc tgg aag cag tcc acc cag cgc ctg cgc agg 384
Phe Phe liln Tyr Asp Thr Trp Lys Gla 8er Thr Aln Arg Lsu Arg Arg
115 1Z0 1~5
ggc ctg cct gcc ctc ctg cgt gcc cgc cgg ggt cac gtg ctc gcc sag 43~
aly Leu Pro Ala Leu Leu Arg Ala Arg Arg aly 81s Val Leu Ala Lys
130 135 140
gag ctc gag gcg ttc agg gag gce aaa cgt cac cgt ccc ctg alt get 480
Qlu Leu Olu Ala Phe Arg Giu Ala Lye Arg 81s Arg Pro Leu Ile Ala
145 150 155 160
cta ccc acc caa gac ccc gcc cac Qgg gga gca cc~c cca gag atg gcc 5Z8
Leu Pro Thz ala Asp Pro Ala 81s fly Qly Ala Pro pro G1u Ilst Ala
lb5 170 175
agc aat cgg aag tga 543
8er Asa Arg Lys
180
<Z10>6
<Z11>1B0
<212>PRT
<a13>80~o sapiens
CA 02341314 2001-12-05
W4 00/I4~7I p'C1'1US99/2020~
<400> 6
bet Gly Ile Pro lsat Oly Lys Ssx 1ht Leu Val Leu Leu Thr Phe Leu
1 5 10 15
Ala Phe Ala Ser Cys Cys =le Ala 711a T'yr Arg Pro 8er alu Thr Leu
20 Z5 30
Cys Gly Gly Glu Leu Val llsp Thr Leu Glla Pbe Val Cue Gly flap ~
35 40 45
Gly phi Tyr Phe Ssr Arg Pro 111a Ssr Arg Val Ser Arg Arg Ser Arg
50 55 60
Oily I1e val Giu Glu Cys Cys Phe Arg Sar Cys Asp Lsu Ala Leu Lau
65 70 75 80
Glu Thr Tyr Cps Ala Thr Pro Aia Irys 8er Glu Arp Asp Val Ser Thr
85 90 95
Pro Pro Thr Val Leu Pro Asp Asn Phe Pro ArQ Tyr Pro Val Gly Lys
100 105 110
Phe Phe Ola Tyr Asp Thr Trp Lys Ola Ser Thr Gln Asg Leu Arg ArQ
115 iS0 1~5
Gly Leu Pro Ala Lsu Leu Arg 11"7.a ~ ArQ Gly 8is Val Lsu Ala Lys
130 195 140
Glu Lau Glu Ala Phe Arg Olu 111a Lys Arg 8is Arg Pro Leu =le Ala
145 150 155 160
Leu pro Thr Gla Asp Pro Ala 81s Gly A1y Ala Pro Pro Giu taet Ala
165 170 175
Ser Asa Arg Lys
180
<Z10> 7
<211> 717
<~12> aNA
<213> Artificial Sequeace
<ZZO>
<Z21> CD8
<2Z2> (1)..(717)
CA 02341314 2001-12-05
WO 00/14271 PCT/US99/Z0207
<ZZO>
<Z23> Description of Artificial Sequences humanised
green fluorescent protein eDI~A
<400> 7
atg agc aag ggc gag gaa ctg ttc act ggc gtg gtc cca aft ctc gtg ~8
Met Ser Lys Gly Glu Glu Leu Phe Thr Oly Val Val Pro Ile Lsu Val
1 5 10 15
gaa ctg get ggc get gtg eat ggg cac aaa ttt tat gte agc gga gag 96
Glu Leu Asp Giy Asp Val Asn Gly 81s Lye Phs Ser Val Ser Aly Glu
ZO 25 30
ggt gaa ggt get gcc sca tac gga aag ctc acc ctg aaa ttc atc tgc 144
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
35 90 45
acc act gga aag cte cct gtg eca tgg cca ace etg gte act ace tte 192
Thr Thr Gly Lys Leu Pro Val pro Trp Pro Thr Leu Val Thr Thr Phe
50 55 60
tct tat gge gtg cag tge ttt tcc age tac eca gac cat atg aag cag 380
Ser Tlrr Gly Val Gln Cys Phe Ser Arg Tyr pro Asp His leaf Lys Gla
65 70 75 80
cat gac ttt tte aag age gcc atg ecc gag ggc tat gtg cag gag age 288
His Asp Phs Phe Lye Ser Ala llat Pro Olu Qly Tyr Val Oln Olu Arg
85 90 95
ace ate ttt tte aas get gac ggg aac tae aag aec age get gaa gtc 336
,, Thr Ile Phe Phe Lys Asp Asp Aly Asn Tyr Lya Thr Arg Ala Glu Val
100 105 110
aag ttc gaa ggt gee ace ctg gtg eat age ate gag ctg aag ggc aft 38~
Lys Pha Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
115 120 1~5
gac ttt aag gag get gga aac att ate gge cac aag etg gaa tae aac 432
Asp Phe Lys Glu Asp Gly Asa Ile Leu Gly His Lys Lsu Glu Tyr Asa
130 135 180
tat aac tcc cac sat gtg tae ate atg gcc gac aag caa aag eat ggc 880
err Asn Ser 81s Asn Val Tyr Ile Met Ala Asp Lys Gia Lys Asn Gly
145 150 155 160
ate aag gtc aac ttc aag ate age cac aac aft gag get gga tcc gtg 528
ile Lys Val Asn Phe Lys ile Arg His Asn Ile Glu Asp Gly Bar Vsl
165 170 175
CA 02341314 2001-12-05
WO 00/14Z'11 PCT/US99/20Z87
cag ctg gcc gac cat tat caa cag aac act cca atc ggc gac ggc cct 576
Gln Leu Ala Asp 81s Tyr Gla Gla Jlsa Thr Pro Ile Gly Asp Gly pro
180 185 190
gtg ctc ctc cca gac aac eat tac ctg tcc acc eag tct gec ctg tet 6Z4
Val Lau Leu Pro flap llsn 81s Tyr Lau 8er Thr Gln Ser 111a Leu Set
195 300 305
aaa gat ccc aac gaa aag aga Qac cac atg gtc ctg ctg gag ttt gtg 693
Lys Asp Hro hsn Glu Lys llrg lisp 8is tt~t Val Lsu Leu Glu Phe Val
310 315 330
ace get get ggg ate aca cat ggc atg gac gag etg tae aag tga 719
Thr Ala Ala Gly ile Thr 8is Gly ltet Asp Glu Leu Tyr Lys
325 330 335
<310> 8
<311> 338
<312> PRT
<313> Artificial Sequence
<400> 8
lcet 8er Lys Gly Glu Glu Lsu phe Thr Gly Val Val pro Ile Leu Val
1 5 10 15
Glu Leu hsp Gly ~lsp Val llsa Gly 8is Lys phe 8sr Val Ser Gly Glu
30 35 30
... O~.y Glu Gay Asp ~11a Thr Tyr Gly Lys Leu Thr ~Leu Lys pha Ila GSrs
35 40 45
Thr Thr Gly Lys Leu Pro Val pro Txp Pro Thr Leu Val Thr Thr Phe
50 55 60
Ser Tyr Giy Val Gla Cys phs Ser llrg Tyr Pro lisp 8is flet Lyr Gln
65 70 95 80
8is hsp phe phe Lys Ser Ala ttat pro G1u Gly Tyr Val Gla Glu llrg
85 90 95
Thr Ile Phe phe Lys lisp Asp Gly 1ua Tyr Lys Thr ~lrg Ala Glu Vai
100 105 110
Lys phe Glu Gly Asp Thr Leu Val Asa J~rg Ila Giu Leu Lys Gly Ile
115 120 i35
CA 02341314 2001-12-05
wo oonaZm Pcrnrs99noZO~
Asp Phe Lys Glu Asp Gly Asa Ile Leu Aly 8is Lys Leu Alu Tyr Aaa
I30 135 140
Tyr Aaa Ser 8is Aaa Val Tyr =le ttat Ala Asp Lys Oln Lys Asa Oly
145 150 155 160
Ile Lys Val Asa Phs Lys =le Arg 8is Asa Ile Olu Asp Aly 8er Dal
lb5 190 175
Gla Leu Ala Asp 8is Tyr Ala Ola Asa Thr Pro ile Oly Asp Oly Pro
180 185 190
Val Leu Leu Pro Asp Asa 8is Tyr Leu Ser Tbr Ola 8er A1a Leu Ser
195 X00 Z05
Lys Asp Pro Asa Glu Lys Arg Asp 8is lfat Val Leu Leu Glu Phe Val
210 Z15 ZZO
Thr Ala Ala Gly Ile Thr 8is Oly llet Asp Olu Lsu Tyr Lye
Z25 Z30 Z35
<Z10>9
<~11>333
<slz>a~A
<Z13>8amao sapieas
<ZZO>
<Zal> cas
<asZ> (1) .. (333)
<400> 9
atg gcc ctg tgg atg cgc ctc ctg ccc ctg atg gcg ctg ctg gcc ctc ~18
Diet Ala Leu Trp llet Arg Lsu Leu Pro Leu Leu Ala Leu Leu Ala Leu
1 5 10 15
tgg gga cct gac cca gcc gca gcc ttt gtg aaa caa cac ctg tgc ggc 96
Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asa Gala 8is Leu Cys Oly
20 Z5 30
tca cac ctg gtg gaa get ctc tac cta gtg tga ggg gaa cga ggc ttc 1~4
8er His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Oly Phe
35 '10 ~5
ttc tac aca ccc aag acc cgc cgg gag gca gag gac ctg csg gtg qgg 19a
Phe.Tyr Thr Pro Lys Thr Arg Arg Glu Ala Olu Asp Leu G1a Vai Gly
50 55 60
CA 02341314 2001-12-05
WO OOJ14Z91 PCTNS99I20Z07
caQ gtg gag ctg gge ggg Qgc cct ggt Qca ggc agc ctg eag cca ttg Z40
Oln Val Olu Lau Oly Oly Oly Pro Oly ,illa Oly Ber Leu 01a pro Leu
85 70 75 80
gcc ctg Qag gQg tcc ctg cag aay agt ggc att gtg gaa aaa tgc tgt Z88
Ala Leu Olu Oly eer i.eu Ola Lya Arg Oly Ile Val 31u Ola C~ra Cys
85 90 95
acc agc atc tpc tcc ctc tac cag ctg Qag aac tac tgc aac taQ 333
Ths 8er I1e Cys Sar Lsu Tyr 01a teu Olu Aaa Tyr Cys llsn
100 105 110
<Z10>10
<Z11>110
<~la>PRT
<a13>80~o aapiena
<<00> 10
let 711a Leu Trp llet Jlrg Leu Lau pro Leu Leu 111a leu Leu 711a Leu
1 S 10 i5
Trp Oly pro leap pro Jlls 111a Ai.a phe Val ~laa Ola 8ie l.eu Cye Oly
ZO Z5 30
8er 8ia teu Val Olu 111a lau Tyr Leu Val CYs Oly Olu Arg Oly phe
35 80 8S
Phe Tyr Thr pro Lya Thr ~ Arg Alu 111a Olu hap Leu Ola Val 01y
50 55 60 ,
Gln VaI 01u Leu Oly Oly Oly Pro Oly ~11a Oly 8er Leu Ola pro Leu
65 70 95 80
l~la Leu Olu Aly 8sr Leu 01a Lya Arg 01y Ile Val 01u Ola Cars Cya
B5 90 9S
Thr Ser Ile Cya 8er Leu Tyr Ola beu 01u llsn Tyr Cya 7lsa
100 105 110
