Note: Descriptions are shown in the official language in which they were submitted.
W094/11512 2~71~ PCr/US93/11000
AI.LERGENIC PROTEINS AND PEPTIDES
FROM JAPANESE CEDAR POLLEN
.
Back~round of the Invention
Genetically predisposed individuals, who make up about 10% of the
population, become hypersensitized (allergic) to antigens from a variety of
~` environmental sources to which they are exposed. Those antigens that can induce
immediate and/or delayed types of hypersensitivity are known as allergens. ~King,
- T.P., Adv. Immunol. 23: 77-105, (1976)). Anaphylaxis or atopy, which includes the
- symptoms of hay fever, asthma, and hives, is one form of immediate allergy. It can
be caused by a variety of atopic allergens, such as products of grasses, trees. weeds,
animal dander, insects, food, drugs, and chemicals.
The antibodies involved in atopic allergy belong primarily to the IgE class of
immunoglobulins. IgE binds to mast cells and basophils. Upon combination of a
- ~ ~; specific allergen with IgE bound to mast cells or basophils, the IgE may be cross-
link~ed on the cell surface, resulting in the physiological effects of IgE-antigen
interaction. These physiological effects include the release of, among other
substances, histamine, serotonin, heparin, a chemotactic factor for eosinophilicleukocytes and/or the leukotrienes, C4, D4, and E4, which cause prolonged
constriction of bronchial smooth muscle cells (Hood, L.E. et al. Immunology (2nded.), The Benjamin/Cumming Publishing Co., Inc. (1984)). These released
substances are the mediators which result in allergic symptoms caused by a
combination of IgE with a specific allergen. Through them. the effects of an
allergen are manifested. Such effects may be systemic or local in nature, depending
on the route by which the antigen entered the body and the pattern of deposition of
IgE on mast cells or basophils. Local manifestations generally occur on epithelial
surfaces at the location at which the allergen entered the body. Systemic effects can ~,
include anaphylaxis (anaphylac~ic shock), which is the result of an IgE-basophilresponse to circulating (intravascular) antigen.
Japanese cedar (Sugi; C yptomeria japonica) pollinosis is one of the most r
important allergic diseases in Japan. The number of patients suffering from thisdisease is on the increase and in some areas, more than 10% of the population are
affected. Treatment of Japanese cedar pollinosis by administration of Japanese cedar
pollen extract to effect hyposensitization to the allergen has been attempted.
Hyposensitization using Japanese cedar pollen extract, however, has drawbacks in
WO 94/l1~12 ~ 1 1 8 71 c~ PCI/US93/11000 ~t
that it can elicit anaphylaxis if high doses are used, whereas when low doses are used
to avoid anaphylaxis, treatment must be continued for several years to build up a
tolerance for the extract.
The major allergen from Japanese cedar pollen has been purified and
s designated as Sugi basic protein (SBP) or Cry j I. This protein is reported to be a
basic protein with a molecular weight of 41-S0 kDa and a pI of 8.8. There appear tO
be multiple isoforms of the allergen, apparently due in part to differential
glycosylation (Yasueda et al. (1983) J. Allergy Clin. Immunol. 71: 77-86; and Taniai
et al . (1988) FEBS Letters 239: 329-332 . The sequence of the first twenty amino
lo acids at the N-terrninal end of Cry j I and a sixteen amino acid internal sequence
have been determined (Taniai supra).
A second allergen has recently been isolated from the pollen of Cryptomeria
japonica (Japanese cedar) (Sakaguchi et al. (1990) Allergy 45:309-312). This
allergen, designated Cry j II, has been reported to have a molecular weight of
s approximately 37 kl~a and 45 kDa when assayed on sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing
conditions, respectively (Sukaguchi et al., supra). Cry j II was found to have no
immunological cross-reactivity with Cly j I (Sakaguchi (1990) supra; Kawashima et
~`:
al. (1992) Int. Arch. Allergy Immunol. 98:110-117). Most patients with Japanese
;~ 20 cedar pollinosis were found to have IgE antibodies to both C~y j I and C~y j II.
- however, 29% of allergic patients had IgE that only reacted with Cry; I and 14% of
~ allergic patients had IgE that only reacted with C~y j II (Sakaguchi (1990) supra).
J Isoelectric focusing of Cry j II indicated that this protein has a pI above 9.5. as
~, compared to pI 8.6-8.8 for C)y; I (Sakaguchi (1990) supra). Further, the reported
2s NH2-terminal sequence for Cry j II, NH2-AlaIleAsnIlePheAsnValGluLysTyr-
COOH, did not match that reported for C~y j I (Sakaguchi (1990) ~E~)
Despite the attention Japanese cedar pollinosis allergens have received,
definition or characteri~ation of the allergens responsible for its adverse effects on
people is far from complete. Current desensitization therapy involves treatment with
pollen extract with its attendant risks of anaphylaxis if high doses of pollen exlract
are administered, or long desensitization times when low doses of pollen extract are ~-
administered~ ,~
J
Sun~nar~ of the Invention
The present invention provides nucleic acid sequences coding for the
Cryptomeria japonica major pollen allergen Cry j II and fragments thereof. The
present invention also provides purified Cly j II and at least one fragment thereof
/094/11512 21'1~713 Pcr/uss3/llooo
produced in a host cell transformed with a nucleic acid sequence coding for C~y j II i
or at least one fragment thereof and fra~ments of C y j II prepared synthetically.
~; ~ As used herein, a fragment of the nucleic acid sequence coding for the entire amin~o
acid sequence of C~y j II refers to a nucleotide sequence having fewer bases than the 1-
nucleotide sequence coding for the entire amino acid sequence of Cry j II and/ormature CrY J; II. Cry j II and fragments thereof are useful for diagnosing, treating~
and preventing Japanese cedar pollinosis. This invention is more particularly
described in the appended claims and is described in its preferred embodiments in
the following description.
Description of the Drawin~s
Fig. la shows an SDS-PAGE (12%) analysis of C~yj II under non-reducing
conditions.
Fig. lb shows an SDS-PAGE (12%) analysis of Cry j II under reducing
conditions.
- Fig~ 2 shows the results of mono S column chromatography of Cry j II eluted
with a step gradient of NaCI in 10mM sodium acetate buffer, pH 5Ø
Fig. 3 shows an SDS-PAGE (12%) of purified subfractions of C~yj II
analyzed under reducing conditions.
20 ~ ~ ~ Fig. 4 shows the nucleic acid sequence (SEQ ID N0: 1) and the deduced
amino acid (SEQ ID N0: 2) coding for C~y j II.
Fig. S shows the deduced amino acid sequence of C~y j II (SEQ ID N0: 2).
Fig. 6 shows the long form (SEQ ID N0: 4) and short forrn (SEQ ID N0: 5)
NH2-terrninii amino acid sequences of C~y j II deterrnined by protein sequence
;25 analysis as discussed in Example 2 aligned with the ten amino acid sequence of C~y
j II (SEQ ID N0: 3) defined by Sakaguchi et al., supra (SEQ ID N0: 6).
Fig. 7 is a graphic representation of the results of a direct ELISA assay
showing the binding response of the monoclonal antibody 4Bl l and seven patients'
(Batch l) plasma IgE to purified Cry j I as the coating antigen.
Fig. 8 is a graphic representation of a direct ELISA assay showing the
- ~ binding response of the monoclonal antibody 4Bl l, and seven patients' (Batch 1)
plasma IgE to purified native Cry j II as the coating antigen. ~-
Fig. 9 is a graphic representation of a direct ELISA assay showing the
~ binding response of the monoclonal antibody, 4Bll, and seven patients' (Batch 1)
- ~ 3S plasma IgE to recombinant Cry j II (rCry j II) as the coating antigen.
Fig. lO is a graphic representation of a direct ELISA assay showing the
binding response of eight patients' (Batch 2) plasma IgE to purified native C~y j E
~:
. : .
'~
~,~ ..... ". . . . .- . .. .. ~.. . . . .
WO 94/l 1512 ~ 1 4 ~ ~ 1 3 4 Pcr/us93/
Fig. 11 is a graphic representation of a direct ELISA assay showing the '
binding response of eight patients' (Batch 2) plasma IgE to purified native Cry j II.
Fig. 12 is a graphic representation of a direct ELISA assay showing the
binding response of eight patients' (Batch 2) plasma IgE to recombinant Cry j II.
s Fig. 13 is a graphic representation of a direct ELISA assay showing the ~,
binding response of eight patients' (Batch 3) plasma IgE to purified natilve Cr),~ j I.
Fig. 14 is a graphic representation of a direct ELISA assay showing the
` binding response of eight patients' (Batch 3) plasma IgE to purified na~ive Crv j II.
Fig. 15 is a graphic representation of a direct ELISA assay showing the
0 binding response of eight patients' (Batch 3) plasma IgE to recombinant Cry j II.
Fig. 16 is a table which summarizes both the MAST scores performed on
patient's plasma samples (Batch 1-3) and the direct ~LISA results shown in Figs. 7-
15; a positive response is indicated by a (+) sign and the number of positive
responses for each antigen is shown at the bottom of each column.
Detailed Description of the Invention
The present invention provides nucleic acid sequences coding for C~ j II, an
allergen found in Japanese cedar pollen. The nucleic acid sequence coding for Cry j
II shown in Fig. 4 (SEQ ID NO: 1) encodes a protein of 514 amino acids. The
deduced C7y j II amino acid sequence is shown in Figs. 4 and 5 (SEQ ID NO: 2) .
Direct protein sequence analysis of native purified Cry j II resulted in two separate
overlapping NH2-termini sequences, designated Long and Short, corresponding
respectively to amino acids 46 through 89 (SEQ ID NO: 4) and 51 through 89 (SEQ
l ID NO: 5) of Figs. 4, 5 and 6. The ten amino acid sequence NH2-AlaIleAsnIlePhe-
2s AsnValGluLysTry-COOH (SEQ ID NO: 6) previously defined by Sakaguchi et al,
su~ra for c~y j II corresponds to amino acids 55 through 64 of Figs. 4 and 6. The
full-length cry j II sequence contains 20 cysteine residues and three potential N-
linked glycosylation sites with the consensus sequence of Asn-Xxx-Ser/Thr.
According to the program contained in PC Gene, Intelligenetics (Mountain View,
CA) the proteins with the NH2-termini defined by the Long and Short forms of C~y j
II would contain 469 and 464 amino acids, respectively, and have predicted
molecular weights of 51.5 kDa (long) and 50.9 kDa (short). The amino acid
sequence representing the long form of C~y j II is encoded by the nucleotide
sequence extending from bases 177-1586 (SEQ ID NO: 7) as shown in Fig. 4, and
the arnino acid sequence representing the short form of Cry j II is encoded by the
nucleotide sequence extending from 192-1586 (SEQ ID NO: 8) as shown in Fig. 4.
A host cell transformed with a vector containing the cDNA insert coding for full-
"~
: . . . .
~;-r~t ~ 1 4 ~ 7 1 3
WO 94/11512 ~ ~Cr/uss3/1 1000 1 --
i --
length C~y j II has been deposited with the American Type Culture Collection,
ATCC No. 69105 .
Fragments of the nucleic acid sequence coding for fragments of Cry j II are~
also within the scope of the invention. Fragments within the scope of the invention
mclude those coding for parts of Cry j II which induce an immune respanse in 1.
!- mammals, preferably humans, such as stimulation of minimal amounts of IgE;
;~ bindlng of IgE; eliciting the production of IgG and IgM antibodies; or the eliciting
~t: of a T cell response such as proliferation and/or Iymphokine secretion and/or the
1 ~ induction of T cell anergy. The foregoing fragments of Cry j II are referred to herein
i ~lo as antigenic fragments. Fragments within the scope of the invention also include
those capable of hybridizing with nucleic acid from other plant species for use in
screening protocols to detect allergens that are cross-reactive with Cry j II. As used
herein, a fragment of the nucleic acid sequence coding for Cry j II refers to a
nucleotide sequence having fewer bases than the nucleotide sequence coding ~or the
entire amino acid sequence of C~y j II and/or mature Cry j II. Generally, the nucleic
acid sequence coding for the fragment or fragments of Cry j II will be selected from
the bases coding for the mature protein, however, in some instances it may be
desirable to select all or a part of a fragment or fragments from the leader sequence
; ponion of the nucleic acid sequence of the invention. The nucleic acid sequence of
t he invention may also contain linker sequences, modified restriction endonuclease
sites and other sequences useful for cloning, expression or purification of C~y j II or
fragments thereof.
A nucleic acid sequence coding for Cry j II may be obtained from
Cryptomeria japonica plants. Applicants have found that fresh pollen and staminate
2s cones are a good source of Cry j II mRNA. It may also be possible tO obtain the
nucleic acid sequence coding for Cry j II from genomic DNA. C~yptomeria
japonica is a well-known species of cedar, and plant material may be obtained from
wild, cultivated, or ornamental plants. The nucleic acid sequence coding for Cry j II
~ may be obtained using the method disclosed herein or any other suitable techniques
for isolation and cloning of genes. The nucleic acid sequence of the invention may
be DNA or RNA.
The present invention provides expression vectors and host cells transformed
to express the nucleic acid sequences of the invention. Nucleic acid coding for Cry j
II, or at least one fragment thereof may be expressed in bacterial cells such as E.
3s coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster
ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other
expression control elements may be found in Sambrook et al. Molecular Cloning: A
. .
~1~8713
wo s4t l l 512 PCr/ US93/ 11000
Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, New York (1989). Other suitable expression vectors, promoters,
enhancers, and other expression elements are known to those skilled in the art.
Expression in mammalian, yeast or insect cells leads to partial or complete
glycosylation of the recombinant material and formation of any inter- or intra-chain
disulfide bonds. ~Suitable vectors for expression in yeast include ~epSecl (Baldari et
al. (1987) Embo J. 6: 229-234); pMFa (Kurjan and Herskowitz (1982~ Cell 30: 933-943); JRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (lnvitrogen
Corporation, San Diego, CA). These vectors are freely available. Baculovirus and0 mammalian expression systems are also available. For example, a baculovirus
system is commercially available (PharMingen, San Diego, CA) for expression in
insect cells while the pMSG vector is commerically available (Pharmacia,
~, Piscataway, NJ) for expression in mammalian cells.
For expression in E. coli. suitable expression vectors include, among others,
pTRC (Amann et al. (1988) Gene 69: 301-315); pGEX (Amrad Corp., Melbourne,
Australia); pMAL (N.E. Biolabs, Beverly, MA); pRlT5 (Pharmacia, Piscataway,
NJ); pET-lld (Novagen, Madison, WI) Jameel et al., (1990) J. Virol. 64:3963-
3966; and pSEM (Knapp et al. (1990) BioTechniques 8: 280-281). The use of
pTRC, and pET-l ld, for example, will lead to the expression of unfused protein.The use of pMAL, pRIT5 pSEM and pGEX will lead to the expression of allergen
fused to maltose E binding protein (pMAL), protein A (pRIT5), truncated B-
galactosidase (PSEM), or glutathione S-transferase (pGEX). When Cry j II,
fragment, or fragments thereof is expressed as a fusion protein, it is particularly
advantageous to introduce an enzymatic cleavage site at the fusion junction between
the carrier protein and Cly j II or fragment thereof. C~y j II or fragment thereof
may then be recovered from the fusion protein through enzymatic cleavage at the
enzymatic site and biochemical purification using conventional techniques for
purification of proteins and peptides. Suitable enzymatic cleavage sites include those
for blood clotting Factor Xa or thrombin for which the appropriate enzymes and
protocols for cleavage are commercially available from for example Sigma Chemical
Company, St. Louis, MO and N.E. Biolabs, Beverly, MA. The different vectors
also have different promoter regions allowing constitutive or inducible expression
with, for example, IPTG induction (PRTC, Amann et al., (1988) supra; pET-lld,
Novagen, Madison, WI) or temperature induction (pRIT5, Pharmacia, Piscataway,
3s NJ) . It may also be appropriate to express recombinant C~y j II in different E. coli
hosts that have an aleered capacity to degrade recombinantly expressed proleins (e.g.
U.S. patent 4,758,512). Alternatively, it may be advantageous to alter the nucleic
WO 94/11512 2 1 4 8 7 1 3 PC~`~US93/t 1000 ~;
acid sequence to use codons preferentially utilized by E. coli, where such nucleic
acid alteration would not affect the amino acid sequence of the expressed protein.
Host cells can be transformed to express the nucleic- acid sequences of the
invention using conventional techniques such as calcium phosphate or calcium
~5 chloride co-precipitation, DEAE-dextran-mediated transfection, or electroporation.
Suitable methods for transforrning the host cells may be found in Sambrook et al.
supra, and other laboratory textbooks. The nucleic acid sequences of the invention
may also be synthesized using standard techniques.
The present invention also provides a method of producing purified Japanese
0~ ~ cedar pollen allergen C~y j II or at least one fragment thereof comprising the steps of
culturing a host cell transformed with a DNA sequence encoding Japanese cedar
pollen allergen C)y j II or at least one fragment thereof in an appropriate medium to
produce a mixture of cells and medium containing said Japanese cedar pollen
allergen Cry j II or at least one fragment thereof; and purifying the mixture toi5 ~ produce substantially pure Japanese cedar pollen allergen Cry j II or at least one
fragment thereof. Host cells transformed with an expression vector containing DNA
coding ~for Cly j II or at least one fragment thereof are cultured in a suitable medium
for the host cell. Cry j II protein and peptides can be purified from cell culture
medium, host cells, or both using techniques known in the art for purifying peptides
and proteins including ion-exchange chromatography, gel filtration chromatography,
ultrafiltration, electrophoresis and immunopurification with antibodies specific for
Cly j II or fragments thereof. The terms isolated and purified are used
interchangeably herein and refer to peptides, protein, protein fragments, and nucleic
acid sequences substantially free of cellular material or culture medium when
produced by recombinant DNA techniques, or chemical precursors~
Cr~ j II protein may also be isolated from lapanese cedar pollen as described
in Exarnple l . C~y j II isolated directly from Japanese cedar pollen is referred to
herein as "purif~ed native" C~y j II. It is preferable that purified native C~y j II of ~
the invention be at least 80% pure, and more preferably at least 90% pure and even '
more preferably be purified to homogeneity (at least 99% pure).
Another aspect of the invention provides preparations comprising lapanese
cedar pollen allergen Cry j II or at least one fragment thereof synthesized in a host
cell transformed with a DNA sequence encoding all or a portion of Japanese cedarpollen allergen Cry j II, or chemically synthesued, and purified Japanese cedar
pollen allergen Cry j II protein, or at least one aMigenic fragment thereof produced ~`
in a host cell transformed with a nucleic acid sequence of the invention, or
chemically synthesized. In preferred embodiments of the invention the Cr~ j II
~__,
WO 94/11512 2 1 1 g 7 1 3 PCr/US93/1 1000 ~;
8 . -
protein is produced in a host cell transformed with the nucleic acid sequence coding
for at least the mature C~y j II protein. t
Fragments of an allergen from Cry j II, eliciting a desired antigenic response ',
(referred to herein as antigenic fragments) are defined herein as any protein fragment '
s or peptide which can be derived from the Cry j II proteins, but does not include the ,.
ten amino acid fragments which extends from amino acid residues 55-64~ as shown
in Figs. 4, 5 and 6, but may include any portion of that ten amino acid fragment in
conjunction with another fragment derived from C)y j Il. Antigenic fragments of
~ ; C~y j Il may be obtained, for example, by screening peptides recombinantly
! ~ o produced from the corresponding fragment of the nucleic acid sequence of the
invention coding for such peptides, or by screening peptides which have been
synthesized chemically using techniques known in the art, or by screening peptides
;~ produced by chemical cleavage of the allergen. The allergen may be arbitrarily
divided into fragments of a desired length with no overlap of the peptides, or
preferably divided into fragments of a desired length with no overlap of the peptides,
or preferably divided into overlapping fragments of a desired length.~ The fragments
. are tested to determine their antigenicity (e.g. the ability of the fragment tO induce
an immune response such as T cell proliferation as discussed in Example 7).
Antigenic fragments may also be predicted using an algorithm such as that
discussed in a paper by Hill et al, ~ournal of Immunology, 147:184-197 (199l).
Algorithms for predicting peptides which elicit T cell activity such as the algorithm
discussed by Hill et al. are based on the protein's sequence wherein certain patterns
within the sequence are likely to bind MHC and therefore may contain T cell
epitopes. The peptides predicted by the algorithm such as Cry j IIA and Cry j IIB
discussed in Example 7 may be produced recombinantly or synthetically and testedfor T cell activity as discussed in Example 7.
If fragments of Japanese cedar pollen allergen, e.g. C)y j Il are to be used fortherapeutic purposes, then the fragments of Japanese cedar pollen allergen which are
capable of eliciting a T cell response such as stimulation (i.e., proliferation or '.
lymphokine secretion) and/or are capable of inducing T cell anergy are particularly
desirable and fragments of Japanese cedar pollen which have min~mal IgE
stimulating activity are also desirable. Additionally, for therapeutic purposes, ~ -
purified Japanese cedar pollen allergens, e.g. Cly j Il, and fragments thereof
preferably do not bind IgE specific for Japanese cedar pollen or bind such IgE to a
substantially lesser extent than the purified native Japanese cedar pollen allergen
binds such IgE. If the purified Japanese cedar pollen allergen or fragment or
fragments thereof bind IgE, it is preferable that such binding does not result in the
;~ W094/11~12 2 1 4 8 ~ 1 3 PCr/US93/11000
9 1:~
release of mediators (e.g. histamines) from mast cells or basophils. Minimal IgEstimulating activity refers to IgE stimulating activity that is less than the amount of
IgE production stimulated by the native Cry j II protein.
"r~ Isolated antigenic fragments or peptides of the present invention which have
s ~ T~ cell stimulating activity, and thus comprise at least one T cell epitope are
particularb desirable. T cell epitopes are believed to be involved in initiation and
"`'t~ perpetuation of the imrnune response to a protein allergen which is responsible for
the ~linicàl~symptoms of allergy. These T cell epitopes are thought to trigger early
` ~ events ~at the level of the T helper cell by binding to an appropriate HLA molecule
~l0 ~ ~ on the surface of an antigen presenting cell and stimulating the relevant T cell
subpopulatlon. These events lead to T cell proliferation, Iymphokine secretion, local
inflammatory reactions, recruitrnent of additional immune cells to the site, andactivation of the B cell cascade leading to production of antibodies. One isotype of
- ~ ~ these~ antibodies, IgE, is fundamentally important to the development of allergic
~15~ symptoms and its production is influenced early in the cascade of events, at the level
of ~the T:helper cell, by the nature of the Iymphokines secreted. An epitope is the
; ~ basic~elem~nt or smallest unit of recognition by a receptor, particularly
immunoglobulins, histocompatibility antigens and T cell receptors, where the epitope
comprises ~amino acids essential to receptor recognition. Amino acid sequences
20 ~ which mimic those of the epitopes particularly T cell epitopes and which modify the
allergic response to protein allergens including those capable of down regulating
allergic response to Cry j II, are within the scope of this invention.
As discussed in Example 7, human T cell stimulating activity can be tested by
culturing T cells obtained from an individual sensitive to Japanese cedar pollenS ~ allergen, (i.e., an individual who has an IgE mediated immune response to Japanese
cedar~ pollen allergen) with a peptide derived from the allergen and determining. ~ whether proliferation of T cells occurs in response to the peptide as measured, e.g., it
by cellular uptake of tritiated thymidine. Stimulation indices for responses by T t
cells to peptides can be c?lculated as the maximum CPM in response to a peptide
divided by the control CPM. A stimulation index (S.I.) equal to or greater than two
times the background level is considered "positive" . Positive results are used to L `
calculate the mean stimulation index for each peptide tested. Preferred peptides of ~ `
this invention comprise at least one T cell epitope and have a mean T cell stimulation
index of greater than or equal to 2Ø A peptide having a mean T cell stimulation
3S ~ index of greater than or equal to 2.0 is considered useful as a therapeutic agent. As
shown in Fig. 17 Cr~ j Il peptides Cry j IIA and Cry j IIB have mean stirnulation
- ~ indexes of at least two and therefore comprise at least one T cell epitope as
8~ WO 94/1 1512 1 ~ 7 1 3 Pcr/us93/l lO00
3 lo
predicted.
Purified protein allergens from Japanese cedar pollen or preferred antigenic ;
fragments thereof, when administered to a Japanese cedar pollen-sensitive individual,
or an individual allergic to an allergen cross-reactive with Japanese cedar pollen ' l -
5 ~ ` allergen, are capable of modifying the allergic response of the individual to Japanese
cedar poilen or such cross-reactive allergen of the individual, and preferably are
capable ~of modifying the B-cell response, T-cell response or both the B-cell and the
T-cell response of the individual to the allergen. As used herein, modification of
the~allergic response of an individual sensitive to a Japanese cedar pollen allergen
o ~ can be defined as wn-responsiveness or diminution in symptoms to the allergen, as
determined by standard clinical procedures (See e.g. Varney et al, British Medical
Jou~nal, 302:265-269 (1990)) including dirninution in Japanese cedarpollen
induced asthmatic symptoms. As referred to herein, a diminution in symptoms
includes any reduction in allergic response of an individual to the allergen after the
15 ~ individual has completed a treatment regimen with a peptide or protein of the
invention. This diminution may be subjective (i.e. the patient feels more
comforta'ole in the presence of the allergen). Diminution in symptoms can be
deterrnined clinically as well, using standard skin tests as is known in the art.
The purified C~y j II protein or fragments thereof are preferably tested in
20 mammalian models of Japanese cedar pollinosis such as the mouse model disclosed
i n Tamura et al. (1986) Microbiol. Immunol. 30: 883-896, or U.S. patent
4,939,239; or the primate model disclosed in Chiba et al. (1990) Int. Arch. Allergy
mmunol. 93: 83-88. Initial screening for IgE binding to the protein or fragmentsthereof may be perforrned by scratch tests or intradermal skin tests on laboratory
2S ~ animals or human vo!unteers, or in in vitro systems such as RAST
(radioallergosorbent test), RAST inhibition, ELISA assay, radioimmunoassay (RIA),
or histamine release.
Exposure of allergic individuals to purified protein allergens of the present
invention or to the antigenic fragments of the present invention which comprise at
30 least one T cell epitope and are derived from protein allergens may tolerize or
anergize appropriate T cell subpopulations such that they become unresponsive to the
protein allergen and do not participate in stimulating an immune response upon such
exposure. In addition, administration of the protein allergen of the invention or an
antigenic fragment of the preseM invention which comprises at least one T cell
35~ epitope may modify the lymphokine secretion profile as compared with exposure to
the naturally-occurring protein allergen or portion thereof (e.g. result in a decrease
of IL4 and/or an increase in IL-2). Furthermore, exposure to such antigenic
:: .
~ :
_ . . . . . . . . . . . . . . .
~ 214~713
; WO 94/11~12 ~ Pcr/uS93/llOOO
11 f-
.
- ~ fragment or protein allergen may influence T cell subpopulations which norrnally
participate in the response to the allergen such that these T cells are drawn away
¦ ~ from the site(s) of normal exposure to the allergen (e.g., nasal mucosa, skin, and~ !
lung) towards the site(s) of therapeutic administration of the fragment or protein
5; ~ ~ allergen. This redistribution of T cell subpopulations may ameliorate or reduce the
ability of an individual's immune system to stimulate thc usual irmIlune response at
the site of normal exposure to the allergen, resulting in a dirnunution in allergic
symptoms.
The isolated Cry j II protein, and fragments or portions derived therefrom can
10 ~ ~ be used in methods of diagnosing, treating and preventing allergic reactions to
Japanese cedar pollen allergen or a cross reactive protein allergen. Thus the present
invention provides therapeutic compositions comprising purified Japanese cedar
`~ ~ pollen allergen Cly j II or at least one fragment thereof produced in a host cell
transformed to express C~y j II or at least one fragment thereof, and a
~15 ~ ~ pharmaceutically acceptable carrier or diluent. The therapeutic compositions of the
mvention may also comprise synthetically prepared Cry j II or at least one fragment
thereof and a pha~naceutically acceptable carrier or diluent. Administration of the
therapeutic compositions of the present invention to an individual to be desensitized
can be carried out using known techniques. C~ j II protein or at least one fragment
thereof may be administered to an individual in combination with, for example, an
appropriate diluent, a carrier and/or an adjuvant. Pharmaceutically acceptable
- ~ diluents include saline and aqueous buffer solutions. Pharrnaceutically acceptable
carriers include polyethylene glycol (Wie et al. (1981) Int. Arch. Allergy Appl.Immunol. 64~ 99) and liposomes (Strejan et al. (1984) J. Neuroimmunol 7: 27).
2 5 For purposes of inducing T cell anergy, the therapeutic composition is preferably
administered in nonimmunogenic form, e.g. it does not contain adjuvant. Such
compositions will generally be administered by injection (subcutaneous, intravenous,
~ . ~
etc.), oral administration, inhalation, transdermal application or rectal
administration. The therapeutic compositions of the invention are administered to
3 0 Japanese cedar pollen-sensitive individuals at dosages and for lengths of time
effective to reduce sensitivity (i.e, reduce the allergic response~ of the individual to
Japanese cedar pollen. Effective amounts of the therapeutic compositions will vary
according to factors such as the degree of sensitivity of the individual to Japanese
; - cedar pollen, the age, sex, and weight of the individual, and the ability of the Cty j t.
II protein or fragment thereof to elicit an antigenic response in the individual.
The Cty j II cDNA (or the mRNA from which it was transcribed) or a
portion thereof can be used to identify similar sequences in any variety or type of
,~
WO 94/1 1512 2 1 ~ 8 7 1 3 ~ PCr/US93/1 1000
12
plant and thus, to identify or "pull out" sequences which have sufficient homology to
hybridize to the Cry j II cDNA or mRNA or portion thereof, for exarnple, DNA
~ ;~ from allergens of Cupressus sempervirens, Juniperus sabinoides etc., under ~ ~
conditions of low stringency. Those sequences which have sufficient homology ~ .
5~ (generally greater than 40%) can be selected for further assessment using the method
described herein Alternatively, high stringency conditiolls can be used. In thismarmer, DNA of the present invention can be used to identify, in other types of
- ~ plants, preferably related families, genera, or species such as Juniperus, or
Cupressus, sequences encoding polypeptides having amino acid sequences similar to
lo~ that of ~Japanese cedar pollen allergen Cry j II, and thus to identify allergens in other
species. Thus, the present invention includes not only Cry j II, but also other
allergens~ encoded~ by DNA which hybridizes to DNA of the present invention. Theinvention further includes previously unidentified isolated allergenic proteins or
fragments thereof that are irnmunologically related to Crv j II or fragments thereof,
15 ~ such~as by antibody cross-reactivity wherein the isolated allergenic proteins or
f~ments~ therèof are capable of binding to antibodies specific for the protein and
peptides of ~the invention, or by T cell cross-reactivity wherein the isolated
al!ergenic proteins or fragments thereof are capable of stimulating T cells specific for
the protein and peptides of this invention.
20 ~ ~`Proteins or peptides~ encoded by the cDNA of the present invention can be
used,~ for example as "purified" allergens. Such purified allergens aro useful in the
standardization of allergen extracts which are key reagents for the diagnosis and
treatment of Jàpanese cedar pollinosis~ Furthermore, by using peptides based on the
nucleic acid sequences of Cry j II, antl-peptide antisera or monoclonal antibodies can
2s ~ be ~made using standard methods. These sera or monoclonal antibodies can be used
to standardize~allergen extracts.
Through use of the peptides and protein of the present invention, preparations
of consistent, well-de~med composition and biological activity can be made and
administered for therapeutic purposes (e.g. to modify the allergic response of a- ~ 30 Japanese cedar sensitive individual to pollen of such trees). Administration of such
peptides or protein may, for example, modify B-cell response to Cry j II allergen,
modify T-cell response to C~y j II allergen or modify both B-cell and T-cell 1 -~
responses. Purified peptides can also be used to study the mechanism of
immunotherapy of C~yptomeria japonica allergy and to design modified derivatives, ~ ~ t
35 ~ ~ or analogues useful in immunotherapy.
Work by others has shown that high doses of allergens generally produce the
best results (i.e., best symptom relief). However, many people are unable to
s'~
-~?~ :
WO 94/1 ISI 2 ~ 1 4 ~ 7 ~ 3 pcrtus93J1 1000
13
tolerate large doses of allergens because of allergic reactions to the allergens.
Modification of naturally-occurring allergens can be designed in such a manner that
modified peptides or modified allergens which have the same or enhanced
therapeutic properties as the corresponding naturally-occurring allergen but have
~: s ~ reduced side effects (especially anaphylactic reactions) can be produced. These can
be, for example, a protein or peptide of the present invention (e.g., one having all or
a portion of the amino acid sequence of Cry j II), or a modified protein or peptide,
or protein or peptide analogue.
It is possible to modify the structure of a protein or peptide of the invention
lo ~ ~ for such purposes as increasing solubility, enhancing therapeutic or preventive
efficacy, or stability (e.g., shelf life ex vivo, and resistance to proteolytic
degradadon in vivo). ~ A modified protein or peptide can be produced in which the
~; amino acid sequence has been altered, such as by amino acid substitution. deletion,
or addition, to modify imrnuno~enicity and/or reduce allergenicity, or to which a
component has been added for the same purpose. For example, the arnino acid
residues essential to T cell epitope function can be determined using known
techniques ~e.g., substitution of each residue and determination of the presence or
absence of T cell re?ctivity).
For example, a peptide can be modified so that it maintains the ability
to induce T cell anergy and bind MHC proteins without the ability to induce a strong
proliferative response or possibly any proliferative response when administered in
unmunogenic forrn. In this instance, critical binding residues for the T cell receptor
can be determined using known techniques (e.g., substitution of each residue anddetermination of the presence or absence of T cell reactivity). Those residues shown
2s to be essential to interact with the T cell receptor can be modified by replacing the
essential amino acid with another, preferably similar amino acid residue (a
. ~.
conservative substitution) whose presence is shown to enhance, diminish but not
eliminate binding to relevant MHC.
Additionally, peptides of the invention can be modified by replacing an
amino acid shown to be essential to interact with the MHC protein complex with
another, preferably sirnilar amino acid residue (conservative substitution) whose
` ~ ~ ~ presence is shown to enhance, diminish but not elirninate or not effect T cell ~r .
activity. In addition, amino acid residues which are not essential for interaction with
the MHC protein complex but which still bind the MHC protein complex can be
3 5 modified by being replaced by another amino acid whose incorporation may
enhance, not effect, or diminish but not eliminate T cell reactivity. Preferred amino
- acid substitutions for non-essential amino acids include, but are not limited to
,;
.,
`'
wos4/ll5l2 21~8713 Pcr/US93/llOOO ~ ``
14
substiNtions with alanine, glutamic acid, or a methyl amino acid.
Another example of a modification of protein or peptides is substitution of
cysteine residues preferably with alanine, serine, threonine, leucine or glutamic acid
to mini nize dimerization via disulfide linkages. Another example of modification of
s the peptides of the invention is by chemical modification of amino acid side chains
or cyclization of the peptide.
In order to enhance stability and/or reactivity, the protein or peptides of the
invention Gan also be modified to incorporate one or more polymorphisms in the
àrnino acid sequence of the protein allergen resulting from natural allelic variation.
o~; Addltionally, D-amino acids, non-natural amino acids or non-amino acid analogues
can~be substituted or added to produce a modified protein or peptide within the scope
of lhis lnvention. Furthermore, proteins or peptides of the present invention can be
modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers
~; (Wie et al. supra) to produce a protein or peptide conjugated with PEG. In addition,
5 ~ PEG~ can be added during chemical synthesis of a protein or peptide of the invention.
M~ifications~ of proteins or peptides or portions thereof can also include reduction/
abklation (Tarr in: Methods of Protein Microcharacterization, J.E. Silver ed.
Hurnana Press, ~Clifton, NJ, pp 155-194 (1986)); acylation (Tarr, supra); chemical
coupling to an appropriate`carrier (Mishell and Shiigi, eds, Selected Methods inCellular Immunology, WH Freeman, San Francisco, CA (1980); U.S. Patent
4,939,239; or mild formalin treatment (Marsh International Archives of Allergy and
Applied :lmmunology, 41:199-215 (1971)).
` To facilitate purification and potentially increase solubility of proteins or
peptldes of the invention, it is possible to add reporter group(s) to the peptide
25~ backbone. For example, poly-histidine can be added to a peptide to purify the
peptide on~immobilized metal ion affinity chromatography (Hochuli, E. et al.,
io/lechnology, 6:1321^1325 (1988)). In addition, specific endoprotease cleavage
sitès can be introduced, if desired, between a reporter group and amino acid
sequences of a peptide to facilitate isolation of peptides free of irrelevant sequences.
In order to successfully desensitize an individual to a protein antigen, it may be
necessary to increase the solubility of a protein or peptide by adding functional
:~
~ ~ ~ groups to the peptide or by not including hydrophobic T cell epitopes or regions
containing hydrophobic epitopes in the peptides or hydrophobic regions of the
protein or peptide.
3S~ To potentially aid proper antigen processing of T cell epitopes within a
peptide, canonical protease sensitive sites can be recombinantly or synthetically
engineered between regions, each comprising at least one T cell epitope. For
,~
,,.,
.','.~-~ ' .
~ !' W094/11512 2I~713 PCI`/US93/11000
1:
example, charged amino acid pairs, SUC~I as KK or RR, can be introduced between
regions within a peptide during recombinant construction of the peptide. The
resulting peptide can be rendered sensitive to cathepsin and/or other trypsin-like
enzymes cleavage to generate portions of the peptide containing one or more T cell
s epitopes. In addition, such charged arnino acid residues can result in an increase in
;- ~ solubility of a peptide.
Site-directed mutagenesis of DNA encoding a peptide or protein of the
invention (e.g. Cry j II or a fragment thereof) can be used to modify the structure of
the peptide or protein by methods known in the art. Such methods may, among
o~ others, include PCR withdegenerate oliganucleotides (Ho et al., Gene, 77:51-59
(1989)) or total synthesis of mutated genes (Hostomsky, Z. et al., Biochem. Biophvs,
Res. Comm., 161:1056-1063 (1989)). Toenhance bacterial expression, the
aforementioned methods can be used in conjunction with other procedures to chan~e
~- the eucaryotic codons in I;)NA constructs encoding protein or peptides of the
l5 ~ invention to ones preferentially used in E. coli, yeast, marnmalian cells, or other
`` ~ eukaryotic cells.
Using the structural information now available, it is possible to design Cr~
II peptides which, when administered to a Japanese cedar pollen sensitive individual
in s~ufficient quantities, will modify the individual's allergic response to Japanese
~20~ cedar~pollen. This can be done, for example, by examining the structure of C~yj II,
producing~peptides (via an expression system, synthetically or otherwise) to be
ex`amined for their ability to influence B-cell and/or T-cell responses in Japanese
cedar pollen sensitive individuals and selecting appropriate peptides which contain
~ epitopes recogr~ized by the cells. It is now also possible to design an agent or a drug
;~ 25 ~ ~ ~ capable of blocking or inhibiting the ability of Japanese cedar pollen allergen to
induce an allergic reaction in Japanese cedar pollen sensitive individuals. Suchagents could be designed, for example, in such a maMer that they would bind to
relevant anti-Cry j II IgEs, thus preventing IgE-allergen binding and subsequent mast
cell degranulation. Alternatively, such agents could bind to cellular components of
the imrnune system, resulting in suppression or desensitization of the allergic
response to Cryptomeria japonica pollen allergens. A non-restrictive exarnple of~;~ ~is is the use of appropriate B- and T-cell epitope peptides, or modifications ~ `
thereof, based on the cDNA/protein structures of the present invention to suppress
the allergic response to Japanese cedar pollen. This can be carried out by defining
35 ~ the strucmres of B- and T-cell epitope peptides which affect B- and T-cell function in
- in vitro studies with blood components from Japanese cedar pollen sensitive
individuals .
:
:
' ~ ! .
WO 94/11512 2 1 4 8 71 3 pcr/us93/11000
16 ~`
Protein, peptides or antibodies of the present invention can also be used for
detecting and diagnosing Japanese cedar pollinosis. For example, this could be done
by combining blood or blood products obtained from an individual to be assessed^ for
sensitivity to Japanese cedar pollen with an isolated antigenic peptide or peptides of
Cly j II, or isolated Cry j II protein, under conditions appropriate for binding of
t components in the blood (e.g., antibodies, T-cells, B-cells) with the peptide(s) or
i! protein and determining the extent to which such binding occurs. Other diagnostic
~- methods for allergic diseases which the protein, peptides or antibodies of the present
invention can be used include radio-allergosorbent test (RAST), paper
j 10 radioimmunosorbent test (PRIST), enzyme linked immunosorbent assay (ELISA),
1 radioimrnunoassays (RIA), immuno-radiometric assays (IRMA), luminescence
¦ immunoassays (LIA), histamine release assays and IgE imrnunoblots.
In another diagnostic test, the presence in individuals of IgE specific
for Cry j II at least one protein allergen and the abilitv of T cells of the individuals to
respond to T cell epitope(s) of C~y j Il protein allergen can be determined by
adminis~ering to the individuals an Irnmediate Type Hypersensitivity test and a
Delayed Type Hypersensitivity test. The individuals are administered an Immediate
Type Hypersensitivity test (see e.g. Immunology (1985) Roitt, I.M., Brostoff, J.,
Male, D.K. (eds), C.V. Mosby Co., Gower Medical Publishing, London, NY, pp.
~ 20 19.2-19.18; pp. 22.1-22.10) utilizing the Cry i II protein allergen or a portion
`-~ ~ thereof, or a modified form of the Cry j Il protein allergen or a portion thereof,
each of which binds IgE specific for the allergen. The same individuals are
administered a Delayed Type Hypersensitivity test prior to, simultaneously with, or
subsequent to administraiton of the Imrnediate Type Hypersensitivity test. Of
course, if the Irnmediate Type Hypersensitivity test is administered prior to the
Delayed Type Hypersensitivity test, the Delayed Type Hypersensitivity test would be
given to those individuals exhibiting a specific Immediate Type Hypersensitivityreaction. The Delayed Type Hypersensitivity test utilizes a modified form of theprotein allergen or a portion thereof, the protein allergen produced recombinantly, or
a recombitope peptide derived from the protein allergen, each of which has human T
cell stimulating activity and each of which does not bind IgE specific for the allergen
in a substantial percentage of the population of individuals sensitive to the allergen 7 "
(e.g., at least about 75 %). Based on the results of the above diagnostic tests, those
individuals found to have both a specific lmmediate Type Hypersensitivity reaction
3s and a specific Delayed Type Hypersensitivity reaction are suitable candidates for
administration of a therapeutically effective amount of a therapeutic composition
The thFrapeutic composition comprises the modified forrn of the protein or portion
WO94tllS12 21 4 ~, ~1 3 pcr/uss3/1looo
17
thereof, the recombinantly produced protein allergen, or the recombitope peptide~
each as used in the Delayed Type Hypersensitivity test, and a pharmaceutically
acceptable carrier or diluent. ~ 3
The present invention also provides a method of producing C~y j II or
fragment thereof comprising culturing a host cell containing an expression vector
which contains DNA encoding all or at least one fiagnient of C~yJ II under
conditions appropriate for expression of C~y j II or at least one fragment. The
expressed product is then récovered, using known techniques. Alternatively, Cry j II
or fragment thereof can be synthesized using known mechanical or chemical
techniques.
;~ The DNA used in any embodiment of this invention can be cDNA obtained
as described herein, or alternatively, can be any oligodeoxynucleotide sequence
having all or a portion of a sequence represented herein, or their functional
equivalents. Such oligodeoxynucleotide sequences can be produced chemically or
ls ~ enzymatically, using known techniques. A functional equivalent of an
oligonucleotide sequence is one which is 1) a sequence capable of hybridizing to a
complemeMary oligonucleotide to which the sequence (or corresponding sequence
~portions) of Cry j II or fragments thereof hybridizes, or 2) the sequence (or
corresponding sequence portion) complementary to C~y j II? and/or 3) a sequence
which encodes a product (e.g., a polypeptide or peptide) having the same functional
characteristics of the product encoded by the sequence (or correspondin~ sequence
portion) of C~y j II. Whether a functional equivalent must meet one or both criteria
will depend on its use (e.g., if it is to be used only as an oligoprobe, it need meet
only the first or second criteria and if it is to be used to produce a Cty j II allergen,
~25 it need only meet the third criterion).
The invention is further illustrated by the following non-limiting examples.
Example 1
Purification of Native Japanese Cedar Pollen Aller~en (CrY i II) :
The following purification of native C~y j II from Japanese cedar pollen was ~,
modified from previously published reports (Yasueda et al, J. Allergy Ctin.
Immunol. 71:77 (1983); Sukaguchi et al., Allergy, 45:309 (l990)).
100g of Japanese cedar pollen obtained from Japan (Hollister-Stier, Spokane,
WA) was defatted in lL diethyl ether three times, the pollen was collected afterfiltration and the ether was dried off in a vacuum.
The defatted pollen was extracted at 4C overnight in 2L extraction buffer
:
.
~,.. .~ .. , - . . . ~ -
,. .:
WO 94/11512 Pcr/uss3/1looo ~ '~!~ ~,',''
2148713 1~ :
containing 50 mM tris-HCl, pH 7.~, 0.2 M NaCl and protease inhibitors in final
.~; concentrations: soybean trypsin inhibitor (2 !lg/mL), leupeptin (1 ,ug/mL), pepstatin
A (1 ~Lg/mL) and phenyl methyl sulfonyl fluoride (0.17 mg/mL). The insoluble~
material was re-extrated with 1.2L extraction buffer at 4C overnight and both
~,1 s extracts were combined together and depigmented by batch absorption with 'J
Whatman DE-52 (200g dry weight) equilibrated wilh the extraction buffer.
,~ The depigmented material was then fractionated by ammonium sulfate
precipitation at 80% saturation (4C), which removed much of the lower molecularweight material. The resulting pellet was resuspended in 0.4 L of 50 mM Na-
3 lo acetate, pH 5.0 containing protease inhibitors and was dialyzed extensively against
the same buffer.
The sample was further subjected to purification by either one of the two
methods described below.
Method A
The sample was applied to a 100 rnL DEAE cellulose column (Whatman DE-
52) equilibrated at 4C with 50 mM Na-acetate, pH 5.0 with protease inhibitors.
The unbound material (basic proteins) from the DEAE cellulose column was then
applied to a 50 ml cation exchange column (Whatman CM-52) which was
equilibrated with 10 rnM Na-acetate, pH 5.0 at 4C with protease inhibitors. A
linear gradient of 0-0.3 M NaCI was used to elute the proteins. The early fractions
were enriched in C~y j I whereas the later fractions were enriched in Cly j II.
Fractions containing Cr,v j II were pooled and next applied to an 1 mL Mono S HR5/5 column (Pharmacia, Piscataway, NJ) in 10 mM Na-acetate, pH 5.0, and proteinswere eluted with a linear gradient of NaCI at room temperature. Residual Cry j Iwas eluted at -0.2 M NaCI and Cry j II was eluted between 0.3 to 0.4 M NaCI.
The C~ j II peak was pooled and concentrated to twofold by Iyophilization and
subjected to gel filtration chromatography.
The sample was applied to FPLC Superdex 75 16/60 column (Pharmacia,
Piscataway, NJ) in 10 mM acetate buffer, pH 5.0 and 0.15 M NaCI at a flow rate of
30 ml/min. at room temperature. Purified Cr,v j II was recovered in the 35-30 kDregion. Cr,v j II migrated as ~wo broad bands lower than Cry j I under non-reducing
j conditions (Fig. la) but both bands shifted upward and migrated as C~y j I under
'1 reducing condition (Fig. lb) when analyzed by silver-stained SDS-PAGE. This
3s highly puri~led C~y j II still contained a small amount (~5 %) of Cry j I as detected by
Western blot using MAb CBF2, which has been shown to bind to C~y j I and by N-
terminal protein sequencing. This C~y j II preparation was used to generate primary
;~, . ~ ... . . .;
WO94/11512 21'1~ 713 pcr/us93/llooo
19 !`
protein sequence of Cry J II as described below.
Method B ~ j
The dialyzed sample from the amrnonium sulfate precipitation was applied at ~-
1 ml/min to an 5.0 ml Q-Sepharose Econapac anion exchange cartridge (BioRad,
Richmond, CA) equilibrated with 50 mM Na-acetate, pH 5.0 with protease
inhibitors at 4C. Elution was performed with the above buffer containing 0.5 M
NaCI. The basic unbound material was then applied to a 5.0 ml CM-Sepharose
Econopac cation exchange cartridge (BioRad, Richmond, CA) equilibrated in 50 rnM~10 `~ ~ sodium~acetate pH 5.0 with protease inhibitors. Basic proteins were eluted with a
linear gradient up to 0.1 M sodium phosphate pH 7.0, 0.3 M NaCI at 1 ml/min at
4C. A CtyJ II -enriched peak was collected late in the gradient and further
purified by gel filtration chromatography.
FPLC gel filtration was performed using a 320 mL Superdex 75 26/60
15~ (Pharmacia, Piscataway, NJ) column at 0.5 ml/min in 20 mM sodium acetate, pH
5.0, in~the presence of 0.15 M NaCI. The major peak containing mostly Cry j II
eluted between 160 and 190 ml. Contaminating Cry j I was next removed by FPLC
using a 1.0 ml Mono S 515 (Pharmacia, Piscataway, NJ) cation exchaIlge column
; equilibrated with 10 mM sodium acetate pH 5Ø A stepwise gradient of 0^1 M
~o ~ NaCl was~utilized by~holding isocratically at 0.2 M, 0.3 M, 0.4 M and 1 M salt
concenttation.
Multiple peaks (up to nine peaks) were obtained (Fig. 2) and analyzed by
silver stained SDS-PAGE under reducing conditions (Fig 3). Cry j I with a
~ reported pI of 8.6-8.9 (Yasueda et al, J. Allergy Clin. Immunol., vol. 17 (1983)),
25 ~ ~eluted in the earlier peaks and displayed a molecular weight of about 40 kD. Cry j II
was purified to homogeneity as two bands (Fig. 3) and eluted in the later multiple
peaks, suggesting the existence of isofor ns. ELlSA analysis using the mouse
monoclonal 8Bl I IgG antibody which was raised against biochemically purified Cry
~ j I confirmed the absence of Cry j I in these purified Cry j II preparation. This
¦ 30 purified C~ j Il was used in the human IgE reactivity studies (Example 6).
Phvsical properties of Cns j II ~`~
The physiochemical properties of Cr~ j II were studied and sumrnarized as
¦ ~ ~ below. Under non-reducing SDS-PAGE conditions Cr~ j II consists of two bands
with molecular weights ranged 34000-32000. The molecular weights of both bands
~- are shifted higher to about 38-36 kD under reducing conditions (Fig. lb). This shift
in SDS-polyacrylamide gel has also been observed by others (Sakaguchi et al,
Wo 94/l 1512 2 1 4 ~ 7 1 3 Pcr/uss3/l 1000 `!` ~
Aller~45:309-312 (1990)). These results suggest that intra-disulfide bonds are
probably present in the protein, and it is supported by the present findings that
cloned Cry j II contains 20 cysteines deduced from the nucleotide sequence (Example
~ 3). The pI of Cry j II estimated from IEF gel is about 10. The purifled Cry j II
- ~ ~ s binds human IgE of some allergic patients.
The two moleeular weight bands of Cr~ j II wele ~eparated on a 12~ SDS-
polyacrylamide gel and was then electroblotted onto PVDF membrane (Applied
Biosystems, Foster City, CA). The blot was stained with coomassie brilliant blueand was cut and subjected to N-terrninal amino acid sequencing. (Example 2). Theo results showed that the upper and lower molecular weight bands had identical N-
terminal sequences except the lower molecular weight band missed the first five
arnino acids. The estimated molecular weight of the upper band based on the cDNAsequence is about 52,000, which is significantly higher than the molecular weight
estimated from SDS-polyacrylamide gel either in the presence or absence of reducing
ls ~ ~ reagent. It is also higher than that obtained from gel filtration and preliminary mass
spectroscopy analysis. These are several possibilities to account for this difference.
One possibility is that C~y j II protein is processed. It is probable that the N-
terminal and C-terminal of the protein are cleaved. It is not clear at the present time
whether this processing occurs in the cell or due to proteolysis during purification
even though four different protease inhibitors were added in most of the purification
steps. Nevertheless, the two N-terminal sequences obtained from the purified Cry j
II (Example 2) also contained the N-terminal sequence (10 amino acid) published by
Sakaguchi et al (Allergy, 45:309-312(1990)) suggesting that the N-terminal of C~II is probably hydrolyzed. Since Sakaguchi et al. (supra), did not use any protease
inhibitors in their purification, a higher degree of hydrolysis might have occurred.
This could explain why the N-terminal amino acid sequence that Sakaguchi et al.
obtained was downstream of the N-terminal sequences as discussed in Example 2.
Another approach which may be used to purify native C~ j II or recombinant
Cry j II is in~nunoaf~mity chromatography. This technique provides a very selective
protein purification due to the speci~lcity of the interaction between monoclonal
antibodies and antigen. Murine polyclonal and monoclonal antibodies are generated
against purified C~y j II. These antibodies are used for purification,
characterization, analysis and diagnosis of the allergen C~y j II. `
: :
Example 2
Protein Sequencing of Purified CJY j II
C.Yi II protein was isolated as in Exarnple l . The doublet band shown on
~: '
~ WO 94/11512 2 1 4 ~ 7 1 3 PCr/USs3/1 1000
21
SDS-PAGE (Fig. la) was electroblotted onto ProBlott (Applied Biosystems, Foster
City, CA). Sequencing was performed with the Beckman/Porton Microsequencer
(model LF3000, Beckman Instruments, Carlsbad, CA), a Programmable Solvent
Module (Beckman System Gold Model 126, Beckman Instuments, Carlsbad, CA)
and a Diode Array Detector Module for PTH-amino acid detection (Beckman System
Gold Model 168, Beckman Inslruments, Carlsbad, CA) foliowing manufacturers
specifications.
A~single N-terrninal sequence analysis of the upper doublet band and multiple
N-tèrminal sequence analyses of the lower doublet band showed that both bands
o ~ contained two N-termini, designated "long" and "short". The lower doublet band
contained~approximately 3.3 picomoles of the long form and 8.3 picomoles of the
shon~form. This difference in yields was sufficient to make sequence assignmentsaccording to the quantitation at each sequencer cycle. The upper doublet band
contained approximatelyl8.3 picomoles of both sequences. The revealed long
,~ s- ~ sequence~wasNH2-RKVEHSRHDAINIFNVEKYGAVGDGKH-DCTEAFSTAW(Q)
) ( ) KNP ( ) -COOH, (SEQ ID NO: 4) where (Q) indicates a tentative
i dent~ication of glutarnine at position 38 and () indicated unknown residues atpositions 39~1 and 45. The revealed "short" sequence was NH2-
; SRHDA:tNIFNVE~YGAVGDGKHDCTEAFSTAWS-COOH (SEQ ID NO: 5).
Thus ~the ~ long Cry j II sequence had five additional amino terminal residues than the
short~for n~and the sequence of the short form exactly matched that of the long form.
In addition,~both the long and short forms of Cry j II contained the ten amino acids,
NH2-AINIFNVEKY-COOH (SEQ ID NO 6), previously described for Crv j II
(Sakaguchi et al. 1990t ~). The previously published ten amino acids
~: 25 ~ (Sal~aguchi et al. 1990, suora) correspond to amino acids ten through 19 of the long
form described above.
` Example3
; , ~ Extraction of RNA From Japanese Cedar Pollen and Staminate Cones and '.
Clonin~ of Crv iII
Fresh pollen and staminate cone samples, collected from a single
~yptomena japonica (Japanese Cedar) tree at the Arnold Arboretum (Boston, MA),
were frozen ~mmediately on dry ice. RNA was prepared from 500 mg of each
; sample, essentially as described by Frankis and Mascarhenas (1980) Ann. Bot~ 45:
595-599. The samples were ground by mortar and pestle on dry ice and suspended
in 5 ml of 50 mM Tris pH 9.0 with 0.2 M NaCI, 1 mM EDTA, 0.1~ SDS that had
been treated overnight with 0.1~ diethyl pyrocarbonate (DEPC). After five
. ~ .
':
WO 94/~ 1512 2 1 4 ~ 7 1 3 Pcr/uS93/~ lOoo
22
extractions with phenol/chloroform/isoamyl alcohol (mixed 25:24:1), the RNA was
precipitated from the aqueous phase with 0.1 volume 3M sodium acetate and 2
volumes ethanol. The pellets were recovered by centrifugation, resuspended in 2~ml
dH2O and heated to 65C for 5 minutes. Two ml 4M lithium chloride was added to
s the preparation and the RNA was precipitated overnight at 0C. The RNA pellets .`
were rècovered by centrifugation, resuspended in 1 ml dH2O~ and again preeipitated
with 3M sodium acetate and ethanol on dry ice for one hour. The final pellet waswashed with 70% ethanol, air dried and resuspended in 100 ~I DEPC-treated dH2O
;~ ~ and stored at -80C.
lo ~ Double stranded cDNA was synthesized from 4 ~g pollen RNA or 8 ,ug
flowerhead RNA using a commercially available kit (cDNA Synthesis System kit,
BRL, Gaithersburg, MD). The double-stranded cDNA was phenol extracted,
ethanol precipitated, blunted with T4 DNA polymerase (Promega, Madison, WI),
and then ligated to ethanol precipitated, self annealed, AT and AL oligonucleotides
for use in a modified Anchored PCR reaction, according to the method of Rafnar et
al. (1990) J. Biol. Chem. 266: 1229-1236; Frohman et al. (1990) Proc. Natl.
Acad~. Sci. USA 85: 8998-9002; and Roux et al. (1990) BioTech. 8: 48-57.
~; Oligonucleotide AT has the sequence (SEQ ID NO: 10)
5'-GGGTCTAGAGGTACCG-TCCGTCCGATCGATCATT-3' (Rafnar et al.
- 20 su~ra). Oligonucleotide AL has the sequence (SEQ ID NO: 11)
5'-AATGATCGATGCT (Rafnar et al. supra).
d ~ -~ The first attempts at amplifying the amino terminus of Cry j II from the
linkered cDNA (2 ~l of a 20 ~l reaction) was made using the degenerate.
oligonucleotide CP-11 and oligonucleotide AP. CP-11 has the sequence (SEQ ID
- ~ 25 NO: 12) 5'-ATACTTCTCIACGI~GAA-3', wherein A at positon 1 can be G, C at
position 4 can be T, C at position 7 can be T, I at position 10 is inosine to reduce
degeneracy (Knoth et al. (1988) Nucleic Acids Res. I6: 10932), G at position 13 can
be A, and G at position 16 can be A). AP, which has the sequence (SEQ ID NO:
13) 5'-GGGTCTAGAGGTA-CCGTCCG-3', corresponds to nucleotides 1 through
20 of the oligonucleotide AT. CP-11 is the degenerate oligonucleotide sequence that
is complementary to the coding strand sequence substantially encoding amino acids
PheAsnValGluLysTyr (SEQ ID NO: 14) (amino acids 59 to 64 of Fig. 4), which ~:
correspond to the carboxy terminus of the previously published Cry j II sequence(Sakaguchi et al., supra) shown in Fig. 4. All oligonucleotides were synthesized by
Research Genetics Inc., Huntsville, AL.
Polymerase chain reactions (PCR) were carried out using a commercially
available kit (GeneAmp DNA Arnplification kit, Perkin Elmer Cetus, Norwalk, CT)
:; .
:
`` W 0 94/11512 21~1~713 PCr/US93/11000
~ 23 t~
,~ J
whereby 10 ,ul 10x buffer contair~ing dNTPs was mixed with 100 pmoles of each
oligonucleotide, cDNA (3-5 ,ul of a 20 ~l first strand cDNA reaction mix), 0.5 ,ul
Amplitaq DNA polymerase, and distilled water to 100 ~
The samples were amplified with a programmable thermal controller (MJ
s Research, Inc., Cambridge, MA). The first ~ rounds of amplification consisted of
denaturation at 94C for 1 min, annealing of primers to tlle t~mplate at 45C for 1
min, and chain elongation at 72C for 1 min. The final 20 rounds of amplification
consisted of denaturation as above, annealing at 55C for 1 min~ and elongation as
above. The-primary PCR reaction was carried out with 100 pmol each of the
0 oligonucleotides AP and CP-11. Five percent (S ,ul) of this initial amplification was
then used in a secondary amplification with 100 pmoles each of AP and CP-12. CP-12 has the sequence (SEQ ID NO: 15) 5'-CCTGCAGTACTTCT-
CIACGTTGAAIAT-3', wherein C at position 10 can be T, C at position 13 can be
T, I at positions 16 and 25 are inosines to reduce degeneracy as above, G at position
19 can 've A, and G at position 22 can be A. The sequence (SEQ ID NO: 16) 5'-
CCTGCAG-3' (bases 1 through 7 of CP-12) represents a Pst I site added for
cloning pulposes; the remaining degenerate oligonucleotide sequence is
complementary to the coding strand sequence that substantially encodes the amino -
acids IlePheAsnValGluLysTyr (SEQ ID NO: 17) (amino acids 58-64 of Fig. 4).
Ampli~led DNA was recovered by sequential chloroforrn, phenol, and chlorofo~n
extractions, followed by precipitation on dry ice with 0.5 volumes of 7.5M
ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washing `
with 70% ethanol, the DNA was simultaneously digested with Xba I and Pst I in a
50 ~l reaction, precipitated to reduce the volume to 10 ~1, and electrophoresed
through a preparative 2% GTG NuSeive low melt gel (EMC, Rockport, ME). The
appropriate sized DNA area was visualized by ethidium bromide (EtBr) staining,
excised, and ligated into appropriately digested pUC19 for sequencing by the ',
dideoxy chain termination method of Sanger et al. (1977) Proc. Natl. Acad. Sci.
USA 74: 5463-5476) using a cornmercially available sequencing kit (Sequenase kit,
i, .
U.S. Biochemicals, Cleveland, OH). All resultant clones were sequenced, and nonewere found to contain Cry j II sequence. An alternate 2 PCR reaction was
performed with AP and the nested oligonucleotide CP-21. CP-21 has the sequence
(SEQ ID NO: 18) 5'-CCTGCAGTACTTCTCIACGTTGAAGAT-3' wherein C at
position 10 can be T, C at position 13 can be T, I at position 16 is inosine to reduce
degeneracy as above, G at position 19 can be A, G at position 22 can be A, and G at
position 25 can be A or T. The sequence (SEQ ID NO: 16) 5'-CCTGCAG-3' (bases
1 through 7 of CP-21) represent a Pst I site added for cloning purposes; the
WO 94/1 1512 PfCrtUS93/1 1000 ~ i;~`
~l~lg71~ 24 `
remaining degencrate oligonucleotide sequence is the non-coding strand sequence
corresponding to coding strand sequence substantially encoding amino acids
IlePheAsnValGluLysTyr (SEQ ID NO: 17) (amino acids 58 to 64 of Fig. 4).
A primary PCR was also performed on double-stranded~ cered cDNA
s using CP-23D and AP, as above, to attempt to amplify the 3' end of the Cry j II
cDNA. A secondary PCR was performed using 5 % of ~e priinary reaction, using
CP-24D and AP. CP-23D (sequence (SEQ ID NO: 19) 5'-
GCIATTAATATTTTTAA-3', wherein the T at position 6 can be C or A, T at
position 9 can be C, T at position 12 can be C or A, and T at position 15 can be C )
is the coding strand sequence substantially encoding amino acids AlaIleAsnIlePheAsn
(SEQ ID NO: 20) (amino acids 55 to 60 of Fig. 4); CP-24D (SEQ ID NO: 21)
~sequence 5'-GGAATTCCGCIATTAATATTTTTAATGT-3', wnerein the T at
position 14 can be C or A, T at position 17 can be C, T at position 20 can be C or
A, T at position 23 can be C, and T a~ position 26 can be C ) contains the sequence
5'-GGAATTCC-3' (SEQ ID NO: 22) (bases 1 through 8 of CP-24), which
represents an Eco Rl site added for cloning purposes. The remaining degenerate
oligonucleotide sequence of CP-24D substantially encodes amino acids
AlaIleAsnIlePheAsnVal (SEQ ID NO: 23) (amino acids 55 to 61 of Fig. 4). Again,
multiple clones were sequenced, none of which could be identified as Cly j II, and
this approach was not pursued further.
Upon the characterization of novel C~y j II protein sequence data described in
Example 2, new degenerate oligonucleotides for cloning C~y j II were designed and
¦ synthesized. All oligonucleotides mentioned hereafter were synthesized on an ABI
394 DNA/RNA Synthesizer (Applied Biosystems, Foster City, CA), and purified on
2s NAP-10 colurnns (Pharrnacia, Uppsala, Sweden) as per the manufacturers'
instructions. Degenerate oligonucleotide CP-35 was used with AP on the double-
stranded linkered cDNA in a primary PCR reaction carried out as described herein.
CP-35 has the sequence (SEQ ID NO: 24) 5'-GCTTCGGTACAATCATGm-3',
wherein T at position 3 can also be C; G at position 6 can also be A, T or C; A at
position 9 can also be G; A at position 12 can also be G; A at position 15 can be G;
and T at position 18 can also be C; this degenerate oligonucleotide sequence is the
non-coding strand sequence corresponding to coding strand sequence substantiallyencoding amino acids LysHisAspCysThrGluAla of Cry j II (SEQ Il:~ NO: 25) (amino
acids 71 to 77 of Fig. 4). Five percent (5 ,ul) of this initial amplification, designated
JC136, was then used in a secondary amplification with 100 pmoles each of AP anddegenerate Cly j II primer CP-36, an internally nested Cry j II oligonucleotide primer
with the sequence (SEQ ID NO: 26) 5'-
Wos4/llsl2 ~1 18713 pcr/lJs93/llooo
GGCTGCAGGTACAATCATGTTTGCCATC-3' wherein A at position 11 can also
be G; A at position 14 can also be G; A at position 17 can also be G; T at position 20
can also be C; G at position 23 can also be A, T, or C; and A at position 26 can~also
be G. The nucleotides 5'-GGCTGCAG-3' (SEQ ID NO: 27) (bases 1 through 8 of
s CP-36) represent a Pst I restriction site added for cloning purposes. The remaining }
degenerate oligonucleotide sequence of CP-36 is the noll-coding strand sequence
corresponding to coding strand sequence substantially encoding amino acids
AspGlyLysHisAspCysThr of Cry j II (SEQ ID NO: 28) (amino acids 69 to 75 of Fig.
4). The dominant amplified product, designated JC137, was a DNA band of
o approximately 265 base pairs, as visual~zed on an EtBr-stained 2% GTG agarose gel.
Amplified DNA was recovered by sequential chloroform, phenol, and
chloroform extractions, followed by precipitation at -20C with 0.5 volumes of 7~5
ammonium acetate and 1.5 volumes of isopropanol. After precipitation and washingwith 70% ethanol, the DNA was simultaneously digested with X~a I and Pst I in a
15 ~l reaction and electrophoresed through a preparative 2% GTG SeaPlaque low
~; ~ melt gel (FMC, Rockport, ME). The appropriate sized DNA band was visualized
by EtBr staining, excised, and ligated into appropriately digested pUC19 for
sequencing by the dideoxy chain termination method (Sanger et al. (1977) Proc. Natl
Acad Sci. lJSA 74: 5463-5476) using a commercially available sequencing kit
(Sequenase kit, U.S. Biochemicals, Cleveland, OH).
The clones designated pUC19JC137a, pUC19JC137b, and pUC19JC137e
were found to contain sequences encodin~ the amino terminus of Cty j II. All
three clones had identical sequence in their regions of overlap, although all three
clones had different lengths in the S' untranslated region. Clone pUC19JC137b
was the longest clone. The translated sequence of these clones had complete
identity to the disclosed 10 arnino acid sequence of C~y j II (Sakaguchi et al.,supra.), as well as to the Cry j II amino acid sequence described in Example 2. ,
Amino acid numbering is based on the sequence of the full length prote~n; amino
acid 1 corresponds to the initiating methionine (Met) of Cry j II. The position of
the initiating Met was supported by the presence of an upstrèam in-frame-stop
codon and by 78% homology of the surrounding nucleotide sequence with the
plant consensus sequence that encompasses the initiating Met, as reported by
Lutcke et al. (1987) EMBO J. 6:43~8.
The cDNA encoding the remainder of Cry j II gene was cloned from the
linkered cDNA by using oligonucleotides CP-37 (SEQ ID NO: 29) (which has the
sequence 5'-ATGTTGGACAGTGTTGTCGAA-3') and AP in a primary PCR, `-
designated JC138ii. Oligonucleotide CP-37 corresponds to nucleotides 129 to 149 of
W094/11512 21 l~713 PCI`/VS93/11000
26
Fig. 4, and is based on the nucleotide sequence deteImined for the partial Cry j II
clone pUC19JC137b.
A secondary PCR reaction was performed on 5 % of the initial amplification
mixture, with 100 pmoles each of AP and CP-38 (SEQ ID NO: 30) (which has the
sequence 5'-GGGAATTCAGAAAAGTTGAGCATTCTCGT-3'), the nested primer.
The nucleotide sequence (SEQ I~) NO: 31) 5'-GG~AATTC-3' (bases 1 ~hrough 8 of
CP-38) represents an Eco RI restriction site added for cloning purposes. The
remaining oligonucleotide sequence corresponds to nucleotides 177 to 197 of Fig. 4,
and is based on the nucleotide sequence determined for the partial Cry j II clone
; lo pUC19JC137b. The amplified DNA product, designated JC140iii, was purified and
precipitated as above, followed by digestion with Eco RI and Asp 718 and
electrophoresis through a preparative 1 % low melt gel. The dominant DNA band,
which was approximately 1.55 kb in length, was excised and ligated into pUC19 for
sequencing. DNA was sequenced by the dideoxy chain termination method (Sanger
et al. supra) using a commercially available kit (sequenase kit (U.S. Biochemicals,
Cleveland, OH). Both strands were completely sequenced using M13 forward and
reverse primers (N.E. Biolabs, Beverly, MA) and internal sequencing primers CP-
35,CP-38,CP40,CP-41,CP-42,CP-43,CP-44,CP-45,CP-46,CP-47,CP-48,
CP-49,CP-50, and CP-51. CP-40 (SEQ ID NO: 32? has the sequence 5'-
GTTCTTCAATGGGCCATGT-3' and corresponds to nucleotides 359 to 377 of Fig.
4. CP-41 (SEQ ID NO: 33) has the sequence 5'- GTGTTAGGACT-
GTCTCTCGG-3', which is the non-coding strand sequence that corresponds to
nucleotides 720 to 739 of Fig. 4. CP-42 (SEQ ID NO: 35) has the sequence
5'-TGTCCAGGCCAT-GGAATAAG-3', which corresponds to nucleotides 864 to
2s 883 of Fig. 4 except that the first nucleotide was synthesized as a T rather than the
correct G. CP43 has the sequence (SEQ ID NO: 35)5'-
GCCTTACATGGACTGCAACC-3', which is the non-coding strand sequence that
corresponds to nucleotides 1476 to 1495of Fig. 4. CP44 has the sequence (SEQ
ID NO:36)5'-TCCACGGGTCTGATAATCCA-3', which corresponds to
nucleotides 612 to 631ofPig. 4. CP-45has the sequence (SEQ ID NO:37)
S'-AGGCAGGAAGCAATTTT-CCC-3', which is the non-coding strand sequence
that corresponds to nucleotides 1254to1273ofFig. 4. CP-46 has the sequence ~ "
(SEQ ID NO:38)5'-TACTGCACTTCAGCT-TCTGC-3', which corresponds to
nucleotides 1077 to 1096 ofFig. 4. CP-47 has the sequence (SEQ ID N(:): 39)
3S 5'-GGGGGTCTCCGAATTTATCA-3', which is the non-coding strand sequence thatsubstantially corresponds to nucleotides 1039to1058ofFig. 4, except that the fifth
nucleotide of CP-47 was synthesized as a G rather than the correct nucleotide, T.
= t~ 21 4 ~ 7 1 3 Pcr/us93/l lO00 1~ ~
27
CP-48 (SEQ ID NO: 40), which has the sequence 5'-
GGATATTTCAGTGGACACGT-3', corresponds to nucleotides 1290 to 1309 of
Fig. 4. CP-49 (SEQ ID NO: 41) has the sequence 5'-TATTAGAAGACC-
CTGTGCCT-3', which is the non-coding strand sequence that corresponds to
nucleotides 821 to 840 of Fig. 4. CP-50 (SEQ ID NO: 42) has the sequence
5'-CCATGTAAGGCCAAGTTAGT-3', which corresponds to nucleotides 1485 to
1504 of Fig. 4. CP-51 (SEQ ID NO: 43) has the sequence
5'-ACACCTTTACCCATTAGAGT-3', which is the non-coding strand sequence that
~ ~ corresponds to nucleotides 486 to 505 of Fig. 4.
; ~ ~l o Three clones, designated pUC19JC140iiia, pUC19JC140iiid and
pUC19JC140iiie, were subsequently found to contain partial Cry j II sequence. The
sequence of clone pUC19JC140iiid was chosen as the consensus sequence since it
had the longest 3' untranslated region. The sequences of pUC19JC140iiid and
pUC19JC137b were used to construct the composite Cryj II sequence shown in Fig.
4. In this composite, nucleotide 230 is reported as the A found in pUC19JC137b
(also, pUC19JC137a, pUC19JC140iiia and pUC19JC140iiie) not as the G found in
pUC19JC140iiid; however both A and G at nucleotide 230 encode Lys at amino acid
63. The sequence of clone pUC19JC140iiia was identical to that of pUC19JC140iiid- ~ ~ except for the following: pUC19JC140iiia has a T at nucleotide 357 in place of a C
(no predicted change in amino acid 106), has C at nucleotide 754 instead of T
(changes amino acid 238 from Ile to Thr), C at nucleotide 1246 instead of T
(changes amino acid 402 from Leu to Pro), and T at nucleotide 1672 instead of C
(untranslated region). The sequence of clone pUC19JC140iiie was identical to that
of pUC19JC140iiid except for G at nucleotide 794 instead of A (changes amino acid
25 ~ 251 from Ile to Met), and T at nucleotide 357 in place of C (no predicted change in
amino acid 106).
An ear!ier attempt at cloning the JC140iii PCR product using an Eco Rl/Xba
I digest (oligonucleotide AP has both Xba I and Asp 718 restriction enzyme sites)
yielded cDNA that was cut in half due to an internal Xba I restriction site in the Cly `
j II cDNA, giving rise to 800 and 750 bp bands; the 750 bp band was succesfully
cloned into Eco Rl/X~a I digested pUC19 and sequenced. Two 750 bp clones were ~"
- sequenced and found to be the 5' half of the C~y j II molecule: clones pUC19JC140- .
2a and pUC19JC140-2b. Clone pUC19JC140-2a has C for nucloeotide 297 instead
of T (changes amino acid 86 from Cys to Arg) and clone pUC19JC140-2b has G for
nucleotide 753 instead of A (changes amino acid 238 from Ile to Val). Both clonepUC19JC140-2a and clone pUC19JC140-2b have a T at nucleotide 357 in place of C
(no predicted change in amino acid 106).
~'
~.. , .. .. ~ .. . ...
. . - .. . .
WO94/11512 2148~i3 Pcr/US93/
28
Two different PCR amplifications were also sequenced directly to verify the
clonal c,y j II sequence using the Amplitaq Cycle Seq~lencing kit (Perkin Elmer
Cecus, Norwalk, CT). This procedure involves the ~32P}-end-labelling of ~ !
oligonucleotide sequencing primers which are then annealled (1.6 pmoles in l ~1) to i`
s template DNA and elongated with dideoxy NTPs (methodology of Sanger et al.
(1977) Proc. Natl. Acad. Sci. USA 74:5463-5476) in a PCR reaction also containing
4 ,ul 10X Cycling Mix (contains 0.5 U/~l Amplitaq DNA Polymerase), 5 ,ul template
DNA (lO-100 fmoles) and dH20 to 20 ,ul . The dGTP in the termination mixes in
~: this kit have been replaced by 7-deaza-dGTP, which provides increased resolution of
o sequences containing high G+C regions of DNA. The template DNA was a PCR
product that was recovered by sequential chloroform, phenol, and chloroform
extractions, precipitated at -20C with 0.5 volumes of 7.5 ammonium acetate and
1.5 volumes of isopropanol, then electrophoresed through a preparative 1 or 2%
SeaPlaque low melt gel~ (FMC). Appropriate sized DNA bands were visualized by
EtBr staining, excised, and treated with Gelase (Epicentre Technologies, Madison,
WI) to remove the agarose. The DNA was again precipitated, and resuspended in
50 ~I TE (10 mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) containing 20 ~4glml RNAse
(Boehringer Mannheim, Indianapolis, IN). Two secondary amplifications which had
been used to clone Cry j II were repeated, and used as template DNA for PCR cycle
sequencing: JC137ii, the 5 ' end PCR, (amplified from the 1 PCR JCl36 above)
was rearnplified with oligonucleotides AP and CP-36; and JC140ii, the 3' end PCR,
(amplified from the l c PCR JCl38ii above) was reamplified with oligonucleotidesAP and CP-38. Both of the l ~ amplifications used were precipitated,
electrophoresed through a preparative l or 2 % SeaPlaque low melt gel (FMC), and2S the appropriate sized bands were visualized by EtBr staining and excised. Two ,ul of
each l ~ amplification was then used in the corresponding 2 PCR reaction. The 2
PCR product was then prepared as DNA template for PCR cycle sequencing as
described above. The oligonucleotides used as primers in PCR cycle sequencing,
many of which were used to sequence the clones, are as follows: for JC137ii,CP-36
and CP-39(SEQIDNO:44), which has the sequence 5'- ~
CTGTCCAACATAATTTGGGC-3' and is the non-coding strand sequence .
corresponding to nucleotides 120 to 139 ofFig.4. The oligonucleotide primers used .
for sequencing JC140ii were CP-38, CP-40,CP41,CP-42,CP-43,CP-44,CP-45,
CP-46, CP47, CP49, CP-S0, CP-54(SEQIDNO: 45), which has the sequence 5'-
3s CATGGCAGGGTGGTTCAGGC-3', corresponds to nucleotides 985 to 1004 of Fig.
4,CP-55(SEQIDNO: 46), which has ~e sequence
5'-TAGCCCCATTTACGTGCACG-3' and is the non-coding strand sequence that
WO94/11512 214~ 1 13 pcr/us93t1looo ~.
: !:
~l 29
.
corresponds to nucleotides 929 to 948 of Fig. 4. and CP-56 (SEQ ID NO: 47),
which has the sequence 5'-TTGGGGT~GAGGCCTCCGAA-3' and corresponds tO
nucleotides 1437 to 1456 of Fig. 4. The sequence of this full-length PCR cycle
~? sequencing had only 2 nucleotide changes from the composite
pUC19JC137b/pUC19JC140iiid Cly j Il sequence shown in Figure 4, neither of
which lead tO an amino acid change. There was a T instead of ~ at nucleotide 357(no predicted change in amino acid 106), and a C instead of A at nucleotide 635 (no
,:.,
amino acid change).
The nucleotide and predicted amino acid sequences of Cry j II are shown in
lo Figs. 4 and 5. This is a composite nucleotide sequence from the two overlapping
clones pUC19JC137b and pUC19JC140iiid. Sequencing of multiple independent
clones and cycle sequencing of PCR product confirmed the nucleotide sequence of
Figure 4. There were several nucleotide changes resulting in predicted amino acid
changes, as cited above. However, all nucleotide polymorphisms, with the
exception of the T for C substitition at nucleotide 357, were only observed in single
clones or sequencing reactions. Although T was seen at nucleotide 357 in all clones
except pUCi9JC140iiid, both C and T encode Leu at amino acid 106.
The complete cDNA sequence for C~y j II is composed of 1726 nucleotides,
including 41 nucleotides of 5' untranslated sequence, an open reading frame of 1542
nucleotides starting with the codon for an inidating Met (nucleotides 42-44 of Fig.
4), and a 143 bp 3' untranslated region. There is a consensus polyadenylation signal
sequence in the 3' untranslated region 64 nucleotides 5' to the poly A tail
(nucleotides 1654-1659 of Fig. 4). The position of the initiating Met is confirmed
by the presence of an in-frame upstream stop codon and by 78% homology with the
plant consensus sequence that encompasses the initiating Met (TAAAAUGGC (bases
38 through 46 ~f Fig. 4 (SEQ ID NO: 48)) found in Cry j II compared with the
AACAAUGGC (SEQ ID NO: 49) consensus sequence for plants, Lutcke et al.
(1987) EMBO J. 6: 4348). The open reading frame encodes a deduced protein of
514 amino acids that has complete sequence identity with the published partial -
protein sequence for Cry j II (Sakaguchi et al. ~3apE~), which corresponds to amino
acids 55 through 64 of Fig. 4. The predicted Cly j II protein has 20 Cys, contains
four potential N-linked glycosylation sites corresponding to the consensus sequence ~`
N-X-S/T, has a predicted molecular weight of 56.6 kDa and a predicted pI of 9.08.
Detection of three separate NH2 termini sequences for Cry j II (the long form
and the short form as determined in Example 2 and the NH2 terrninus determined by
Sakaguchi et al., supra, as shown in Fig. 6) may suggest that the amino terminus of
the mature C y j II protein is blocked and that the sequences obtained by sequence
~,~, .=, .,~, . . . .
.~, - . , .
wos4/~s~2 2Iq8713 Pcr/uss3/
':
. ~ analysis of purified protein represent proteolytic cleavage products. As shown in
Fig. 6, the amino acid sequence of the long form of C~y j II begins at amino acid 46
'`~ an,d the amino acid sequence of the short forrn of Cry j II begins at arnino acid 51,~ j-
` ~ and~the NH2-terminal sequence determed by Sakaguchi et al. begins at amino acid
-~ ~ s ~ : 54. ~ It is ~also possible that amino acids 1 to 45 represent the leader/pre-pro position
of ~y j~ll` that is enzymatically cleaved to give a functiorlal~y active protein -
beginning ~at arnino acid 46 of Fig . 4. The sequences beginning at amino acids 51
and~54 represent~breakdo~vn products of the protein beginning at arnino acid 46.There~ is ~a predicted~cleavage site between amino acids 22 and 23 of Fig. 4 using the
'-, lo ~ ~ method o~von~Heijne~(Nucleic Acids Res. (1986) 14:4683-4690). If the mature C~y
D, proteih started' at an~ino acid' 23 in Fig. 4, the protein would be 492 amino acids
`with~`a~predtct~ed~molecular weight of 54.2 kDa and a predicted pE of 9Ø
Searchmg the Swiss-Prot data base with the C~y j II sequence qemonstrated
that ~C~y j II is 43.3 % homologous (33.3 % identical to polygalacturonase of tomato
15 ~ (Lycopets~ n esculentum) and~ 48.4% homologous (32.670 identical) to
polygalacturonase of corn, Zea mays. All nucleotide and amino acid sequence
analyses were performed using PCGENE (Intelligenetics, Mountain View, CA.).
: ~
.~ ~:
~ .
'~ . : ''"
'~
:~
?`
,
,~
`~
_
` ' WO94/11512 214S713 PCr/US93/llOOO
31 :
!
Example 4
Extraction of R~A from Japanese Cedar Pollen Collected in Japan and
Expression of Recombinant Cry j II - ~
Fresh pollen collected from a pool of Cryptomeria japonica (Japanese cedar) c
tre~s in Japan was frozen imrnediately on dry ice. RNA was prepared from 500 mg
of the pollen, essentially as described by Frankis and Mascarenhas Ann. Bot. 45 :595-
599. The samples were ground by mortar and pestle on dry ice and suspended in 5
ml of 50 mM Tris pH 9.0 with 0.2 M NaCI, l mM EDTA, 1% SDS that had been
treated overnight with O. l % DEPC. After five extractions with phenol/chloroforrn
,,:,~ ~,
/ isoamyl alcohol (mixed at 25:24:1), the RNA was precipitated from the aqueous
phase ~with- 0.1 volume 3 M sodium acetate and 2 volumes ethanol. The pellets were
recovered by centrifugation, resuspended in 2 ml dH20 and heated to 65C for 5
minutes. Two ml of 4 M lithium chloride were added to the RNA preparations and
;~ 15 ~ they ~were incubated overnight at 0C. The RNA pellets were recovered by
cennifugation, resuspended in l ml dH20, and again precipitated with 3 M sodium
acetate and ethanol overnight. The final pellets were resuspended in 100 ~41 dH20
and ~stored at -80C.
Double stranded cDNA was synthesized from 8 ~g pollen RNA using the
~ ~ c DNA~Synthesis Systems kit (BRL) with oligo dT priming according to the method
`~ of ~Gubler and Hoffman (1983) Gene 25:263-269. PCRs were carried out using the
~ GeneAmp DNA Amplification kit (Perkin Elmer Cetus) whereby 10 ~l 10x buffer
<~ containing dNTPs was mixed with lO0 pmol each of a sense oligonucleotide and an
anti-sense oligonucleotide, cDNA (lO ~1 of a 400 ~l double stranded cDNA reaction
~-25 ~ mix), 0.5 ~ll Amplitaq DNA polymerase, and distilled water to 100 ,ul.
~- ~ The samples were amplified with a programmable thermal controller from
MJ Research, Inc. (Cambridge, MA). The first S rounds of amplification consistedof denaturation at 94C for 1 min, annealing of primers to the template at 45C for
1 min, and chain elongation at 72C for 1 min. I~e final 20 rounds of amplification
consisted of denaturation as above, annealing at 55C for l min, and elongation as
above.
A new set of primer pairs was synthesized for amplification of a Cry j II ~:
cDNA from the initiating Met to the stop codon. CP-52 (SEQ ID NO: 50) has the
~ sequence 5'- GCCGAATTCATGGCCATGAAATTAATT-3' where the nucleotide
3 5 sequence 5'-GCCGAATTC-3' (SEQ ID NO: 51) (bases l through 9 of CP-52represents an Eco RI restriction site added for cloning purposes, and the remaining
sequence corresponds to nucleotides 42 to 59 of Fig. 4. CP-53 (SEQ ID NO: 52)
. .
WO94/11512 ~148713 pcr/uss3/l1ooo ~ '
has the sequence 5'-CGGGGATCCTCATTATGGATG-GTAGAT-3' where the
nucleotide sequence 5'-CGGGGATCC-3'(SEQID NO:53) (bases 1 through 9 of
CP-53 represents a Bam HI restriction site added for cloning purposes, and the
remaining oligonucleotide sequence of CP-53 is complementary to coding strand
1~ s sequence corresponding to nucleotides 1572 to 1589 of Fig. 4. The PCR reaction
with CP-52 and CP-53 on the double stranded Japanese Cedar pollen cDNA yiclded
a band of approximately 1.55 kb on an EtBr-stained agarose minigel, and was called
JC145. Amplified DNA was recovered by sequential chloroform, phenol, and
chloroforrn extractions, followed by precipitation at -20C with 0.5 volumes of 7.5
amrnonium acetate and 1.5 volumes of isopropanol. After precipitation and washing
with 70% ethanol, the DNA was simultaneously digested with Eco Rl and Bam HI in
a 15 ,ul reaction, and electrophoresed through a preparative 1 % SeaPlaque low melt
gel (FMC). Appropriate sized DNA bands were visualized by EtBr staining,
excised, and ligated into appropriately digested pUC19 for sequencing by the
dideoxy chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA
74:5463-5476) using a cornmercially available sequencing Icit (Sequenase kit, U.S.
Biochemicals, Cleveland, OH).
Clones pUC19JC145a and pUC19JC145b were completely sequenced using
M13 forward and reverse primers (N.E. Biolabs, Beverly, MA) and internal
sequencing primers CP-41, CP-42, CP44, CP-46, and CP-51. The nucleotide and
deduced amino acid sequences of clones pUC19JC145a and pUC19JC145b were
i dentical to the Cry j II sequence of Fig. 4, with the following exceptions. Clone
pUC19JC145a was found to contain a single nucleotide difference from the
previously Icnown C~y j II sequence: it has a C at nucleotide position 1234 of Fig~ 4
rather than the previously described T. This nucleotide change results in a predicted
amino acid change from Ile to Thr at amino acid 398 of the C~y j II protein. Clone
pUC19JC145b has a G at nucleotide position 1088 of Fig. 4 rather than the
previously described A, and an A for a G at nucleotide 1339. The nucleotide change
at 1088 is si!ent and does not result in a predicted amino acid change. The
nucleotide change at position 1339 results in a predicted amino acid change from Ser i;
to Asn at amino acid 433 of the C~y j II protein~ None of these polymorphisms have
yet been confirmed by independently-derived PCR clones or by direct amino acid
sequencing and may be due to the inherent error rate of Taq polymerase t
(appro~cimately 2 x 10~, Saiki et al. (1988) Science 239:487-491). However, suchpolymorphisms in primary nucleotide and amino acid sequences are expected.
¦ Expression of Cry j II was performed as follows. Ten ~g of pUC19JC145b
was digested simultaneously with Eco Rl and Bam HI. The nucleotide insert
.. ..
~h W094/11~12 2 1 4 8 7 i 3 PCI'/US93/11000
33
encoding Cry j II (extending ~rom nucleotide 42 through 1589 of Fig. 4) was
isolated by electrophoresis of this digest through a 1% SeaPlaque low melt agarose :
gel. The insert was then ligated into the appropriately digested expression vector
pET-l ld (Novagen, Madison, WI; Jameel et al. (1990) J. Virol. 64:3963-3966)
-~ S ~ modified to contain a sequence encoding 6 histidines (His 6) immediately 3 ' of the }
ATG ir~itiàtion codon followed by a unique Eco RI endonuclease restriction site. A
second Eco RI endonuclease restriction site in the vector, along with neighboring Cla
I and~ Hind III endonuclease restriction sites, had previously been removed by
digosdon with Eco RI and Hind III, blunting and religation. The histidine (His6)lo ~ sequence was~added for affinity purification of the recombinant protein (Cryj I) on a
Ni2~+~chelating column (Hochuli et al. (1987) J. Ckromatog. 411:177-184; Hochuliet~al.~(1988) Bio/Tech. 6:1321-1325.). A recombinant clone was used to transform`Escherichia coli strain BL21-DE3, which harbors a plasmid that has an isopropyl-13-
D-thiogalactopyranoside (IPTG)-inducible promoter preceding the gene encoding T7~15~ polymerase. Induction with IPTG leads to high levels of T7 polymerase expression,
is~necessary for expression of the recombinant protein in pET-lld. Clone
s6JCI45b.a was confinned to be a Cry j II clone in the correct
reading ~frame for expression by dideoxy se~uencing (Sanger et al. supra) with CP-
39.
- ~2 o Expression of the recombinant protein was examined in an initial small
c ulture. An overnight culture of clone pET-lld~HRhis6JC145b.a was used to
inn~culate 50 ml of media (Brain Heart Inf~sion Mediat Difco) containing ampicillin
(200 ~g/ml), grown to an A600 = 1.0 and then induced with IPTG (1 mMt final
concentration) for 2 hrs. One ml aliquots of the bacteria were collected before and
after inductiont pelleted by centrifugation, and crude cell Iysates prepared by boiling
the pellets for 5 minutes in 50 mM Tris HCI, pH 6.8, 2 mM EDTA, 1% SDSt 1%
; B-mercaptoethanolt 10% glycerol, 0.25% bromophenol blue (Studier et al.t (1990) `~
Methods in Enz~mology 185:6~89). Recombinant protein expression was examined
on a 12% Coomassie blue-stained SDS-PAGE gelt according to the method in
Sambrook et al., supra, on which 25 ~1 of the crude Iysates were loaded. A negative
control consisted of crude Iysate from unLnduced bacteria containing the plasmid ~,:
with Cry j II. There was no notable increase in production of any recombinant E. }
` ~ coli protein in the range of 58 Kd, the size predicted for the recombinant Cry j II
with the His6 leader.
- 3 5 The pET-lld~HRhis6JC145b.a clone was then grown on a larger scale to
examine if there was any recombinant protein being expressed. A 2 ml culmre of
: ~ bacteria containing the recombinant plasmid was grown for 8 hr, then 3 ~I was
"~
,
wo 94/l }512 2 1 4 8 7 1 3 PC-r/Uss3/l lO00
34
spread onto each of ~ (100 x 1~ mm) petri plates with 1.5% agarose in LB medium ,-
(Gibco-BRL, Gaithersburg, MD) containing 200 ~g/ml ampicillin, grown to ¦-
confluence overnight, then scraped into 6 L of liquid media (Brain Heart InfusiQn
media, Difco) containing ampicillin (200 ~g/ml). The culture was grown until theabsorbance at A600 was 1.0, IPTG added (1 mM final concentration), and the
culture grown for an additional 2 hours.
Bacteria were recovered by centrifugation (7,930 xg, 10 min) and lysed in 50
ml of 6M Guanidine-HCI, 0.1M Na2HPO4~ pH 8.0, for 1 hour with vigorous
shaking. Insoluble material was removed by centrifugation (11,000 xg, 10 min, 4o C). The pH of the Iysate was adjusted to pH 8.0, and the Iysate applied tO a 50 ml
Nickel NTA agarose column (Qiagen) that had been equilibrated with 6 M
Guanidine HCl, 100 mM Na2Hpo4~ pH 8Ø The column was sequentially washed
with 6 M Guanidine HCI, 100 rnM Na2HP04, 10 mM Tris-HCl, pH 8.0, then 8 M
urea, 100 mM Na2HPO4, pH 8.0, and finally 8 M urea, 100 mM sodium acetate,
10 mM Tris-HCI, pH 6.3. The column was washed with each buffer until the flow
through had an A280~ 0-05
The recombinant Cry j II protein was eluted with 8 M urea, 100 mM sodium
acetate, 10 rnM Tris-HCl, pH 4.S, and collected in 10 ml aliquots. The protein
concentration of each fraction was determined by A280 and the peak fractions
pooled. An ali~uot of the collected recombinant protein was analyzed on SDS-
PAGE according to the method in Sarnbrook et al. supra.
This 6L prep, JCIIpET-1, yielded 1.5 mg of recombinant Cry j II, which was
resolved into 2 major bands on SDS-PAGE at 58 kDa and 24 kDa. The 58 kDa
band, which represents recombinant Cry j II, was approximately 9-10% of the total
protein as determined by densitometry measurement (Shimadzu Flying Spot Scanner,Shimadzu Scientific Instruments, Inc., Braintree, MA). The 24 kDa band accounts
for about 90% of the total protein and may represent a degradation product of the
recombinant Cry j II or an E. coli contaminant. ,
Another C~y j II expression construct was made by the ligation of the
pUC19JC140iiid Cryj II insert into appropriately digested pETlld~HR (with the 6
histidine leader). The vector was derived from another pETlld~HR construct
whose insert supplied an EcoR I site (at the 5' pET1 ld~HR-insert junction) and an
Asp 718 site (at the 3' end of the insert); the construct was digested with these two `~
enzymes, run on a low melt minigel as above, and the vector recovered as a band in
low melt agarose. The pUC19JC140iiid construct was digested with Eco R I and
Asp 718 to release the Cly j II insert, which was isolated on a low melt minigel and
ligated into the Eco R I/Asp 718 digested pETlld~HR vector prepared above. Five
21~8713
WO94/11~12 pcr/us93/11
clones were found to contain the correct nuc!eotide sequence at the insert/vector 5'
junction, when sequenced by dideoxy sequencing (as above) with CP-39. This new
construct, when expressed, would begin at amino acid 46 of C~y j II as shown in~Figs. 4 and 5. This recombinant protein is designated rCry j II ~46. A 50 ml small '.
scale expression test (as performed above) showed that the expression level of rCry j
1l ~46 from this construct, designated pET11d~HRJC140iiid2, would be much
greater than the initial expression level from pET1 ldAHRJC 145b2 . A 9L prep,
JCIIpET-3, was processed as above, and yielded 200 mg of rCry j Il ~46 at 80%
purity as determined by densitometry of a Coomasie blue stained 12% SDS-PAGE
o gel.
Example S
Northern blot on RNA from Japanese Cedar Pollen Sources
A northern blot analysis was perforrned on the R~A isolated from Japanese
Cedar pollen from both the Arnold Arboretum tree and the pooled trees from Japan.
Using essentially the method of Sambrook, supra, ten ~g of RNA isolated from
Japanese cedar pollen collected from the Arnold Arboretum (Boston, MA) and 15 ,ug
pooled RNA from Japanese cedar pollen collected from trees in Japan were run on a
1.2% agarose gel containing 38% formaldehyde and lX MOPS (20X = 0.4M
MOPS, 0.02M EDTA, 0.1M NaOAc, pH 7.0) solution. The RNA samples (first
precipitated with 1/10 volume sodium acetate, 2 volumes ethanol to reduce volumeand resuspended in 5.5 ~l dH20) were run with 10 ~l formaldehyde/forrnamide
buffer containing loading dyes with 15.5% formaldehyde, 42% formamide, and
1.3X MOPS solution, final concentration. The samples were transferred to
Genescreen Plus (NEN Research Products, Boston, MA) by capillary transfer in 10XSSC (20X = 3M NaCl, 0.3M Sodium Citrate), after which the membrane was
baked 2 hrs at 80C and W irradiated for 3 minutes. Prehybridization of the
membrane was at 60C for 1 hour in 4 ml 0.5M NaPo4 (pH 7.2), lrnM EDTA, 1%
BSA, and 7 % SDS. The antisense probe was synthesized by asymrnetric PCR on the t '
JC145 amplification in low melt agarose (above), where 2 ,ul DNA is amplified with
2 ,ul dNTP mix (0.167mM dATP, 0.167mM dTTP, 0.167mM dGTP, and 0.033mM
dCTP), 2 ,ul 10X PCR buffer, 10 ,ul 32P-dCTP (100 ~Ci; Amersham, Arlington }~
Heights, Il), 1 ~1(100 pmoles) antisense primer CP-53, 0.5 ~l Taq polymerase, and
dH20 to 20 ~l; the 10X PCR buffer, dNTPs and Taq polymerase were from Perkin
Elmer Cetus (Norwalk, CT). Amplification consisted of 30 rounds of denaturation
at 94C for 45 sec, annealing of primer to the template at 60C for 45 sec, and chain `
elongation at 72C for 1 min. The reaction was stopped by addition of 100 ~l TE,
;~ .. , . . ~: .
WO 94/11512 2 1 4 8 7 1 3 pcr/us93/11000
36
- and the probe recovered over a 3cc G-50 spin column (2 ml G-50 Sephadex
[Pharmacia, Uppsala, Sweden] in a 3cc syringe plugged with glass wool,
equilibrated with TE) and counted on a 1500 TriCarb Liquid Scintillation Counter(Packard, Downers Grove, IL). The probe was added to the prehybridizing buffer at
106 cpm/ml and hybridization was carried out at 60C for 16 hrs. Tbe blot was s
washed in high stringency conditions: 3x15 min at 65C with 0.2%SSC/1% SDS,
followed by wrapping in plastic wrap and exposure to film at -80C. A seven hourexposure of this Northern blot analysis revealed a single thick band at approximately
1.7 kb for both RNA collected from the Arboretum tree and the RNA collected fromo~ the pooled trees from Japan. This message is the expected size for Cry j II as
predicted by PCR analysis of the cDNA.
Example 6
Direct bindin~ assav of I~E to Crv i I. Cnl i II and recombinant Crv j II.
Corning assay plates (~25882-96) were coated with Cty j I or C~y j II at 2
g/mL or recombinant Cryj II preparation at 10 ~g/mL (approximately 20% pure)
in a ~volume of 50 ~LL overnight at 4C. The coating antigens were removed and the
wells were blocked with 0.5% gelatin, PVP (polyvinyl pyrolidine) 1 mg/ rnL in
PBS, 200 ~L/well for 2 hours at room temperature. The anti-C~ j I monoclonal
~20 antibody, 4Bl 1, was serially diluted in PBS-Tween 20 starting at a 1:1000 dilution.
The human plasma were serially diluted in PBS-Tween at a starting dilution of 1:2.
For this set 23 plasma samples from patients symptomatic for Japanese cedar pollen
allergy chosen for IgE binding analysis. The first antibody incubation proceededovernight at 4C~ Following three washes with PBS-Tween the second antibodies
2 5 were added (goat anti-mouse Ig or goat anti-human IgE both at 1:2000) andincubated for two hours at room temperature at 100 ~L/well. This solution was
removed and streptavidin-HRPO diluted to 1:10,000, was added at 100~/well. The
color was allowed to develop for 2-5 minutes. The reaction was stopped by the
addition of lOO~L/well of lM phosphoric acid. Plates were read on a Microplate
IL310 Autoreader (Biotek Instruments, Winooski, VT) with a 450nm filter. The
absorbance levels of duplicate wells were averaged. The graphed results (log of the
dilution vs. absorbance) of the ELISA assays are shown in Figs. 7 to 15. The I .
summary of the results are given in Fig~ 16. A positive binding result, indicated by
a plus sign is determined to be a reading of two-fold or greater above background
- 35 (no first antibody) at the second dilution of plasma (1:6).
In Fig. 7 the binding response of the monoclonal antibody, 4B11, and seven
patieMs' (Batch 1) plasma IgE is shown to purified C1y j 1 as the coating antigen.
~ WO 94/1 15 12 2 1 4 ~ 7 1 3 PCI /US93/1 1000
37 ~`
i
The monoclonal antibody, raised against purified Cry j I shows a saturatin~ level of
binding for the whole dilution series. The individual patient samples show a variable - ¦
response of IgE binding to the Cry j I preparation. One patient, #1034, has no
detectable binding to this protein preparation. All the patient samples were obtained
s from individuals claiming to be symptomatic for Japanese cedar pollen allergy and
: ~ the results of their MAST scores are shown in Fig. 16. Fig. 8 is a graph
representing the binding of the same antibody set as in Fig. 7 to purified native C~y j
II. The anti-Cry j I monoclonal antibody, 4B11, is negative on this preparation
demonstrating lack of cross-reactivity between the two allergen antigens. In general,
o there is a lower overall response to this allergenic component of cedar pollen with
more patient samples showing decreased binding. However, patient #1034, that wasnegative on Cry j I shows very strong reactivity to Cry j II. In the last antigen set,
Fig. 9, using recombinant C~y j II (rC~y j II), monoclonal antibody 4B11 reactivity
; is negative and there is further reduc~ion in binding of the human IgE samples
compared to biochemically purified Cry j II. Two of the patients, #1143 and #1146,
are clearly positive for IgE binding to the recombinant form of C~y j II although the
pat~ient that reacted the strongest to biochemically purified form is negative here,
1034. Figs. 10^15 represent the application of the same antigen sets for the direct
binding analysis of the next sixteen patients designated patient Batch 2 and patient
Batch 3 in Figs. 10-15.
The table shown in Fig. 16 sumrnarizes both the MAST scores, performed in
- ~ Japan on the plasma samples before shipment using a commercially available kit, and
the direct ELISA results outlined above. Two patients were negative by the MAST
assay, however, one of these patients, #1143, was positive on all the ELISA
antigens. The number of positive responses for each antigen is shown and this
represents a measure relative allergenicity of the different allergen preparations.
These results demonstrate that C y j II is an allergen as defined by human allergic
patient IgE reactivity and that there are some patients who are not reactive to Cry j I
but are reactive to Gy j II. The frequency of response in this population of patients
is less to Cry j II than to C1y j I.
Example 7
-~ .
Japanese Cedar Pollen Allergic Patient T Cell Studies with CrY i II and Crv j II3S Peptides.
Synthesis of C~y j II Peptides
WO 94/11512 Pcr/uss3/llooo
2148713 38 " ` ~
Japanese cedar pollen C,~y j II peptides designated Cry j IIA Cry j IIB were
synthesized using standard Fmoc/tBoc synthetic chemistry and puri~led by ReversePhase HPLC. The amino acid sequence of peptide Cry J IIA is FTFKVDGIIAAYQ
(SEQ ID NO: 54) which corresponds tO amino acids 116-128 as shown if Figs 4 and
s 5. The amino acid sequence of peptide Cry j IIB is NGYFSGHVIPACKN ~SEQ ID
NO: 55) which corresponds to amino acids 416-429 dS shown in Figs 4 and 5. The
peptide names are consistent throughout.
T Cell Responses to Japanese Cedar Pollen Antigen Peptides
Peripheral blood mononuclear cells (PBMC) were purified by lymphocyte
separation medium (LSM) centrifugation of 60 ml of heparinized blood from one
Japanese cedar pollen-allergic patient who exhibited clinical symptoms of seasonal
rhinitis and was MAST and/or skin test positive for Japanese cedar pollen. Long
term T cell lines were established by stirnulation of 2 X 106 PBL/ml in bulk cultures
of complete medium (RPMI-1640, 2 mM L-glutamine, 100 U/ml
~- penicillinlstreptomycin, sx10-5M 2-mercaptoethanol, and 10 mM HEPES
supplemented with 5 % heat inactivated human AB serum) with 10 ~g/ml of partially
purified native C)y j II for 7 days at 37C in a humidified 5 % C02 incubator to~;~ select for C~y j II reactive T cells. This amount of priming antigen was determined
to be optimal for the activation of T cells from most Japanese cedar pollen allergic
patients. Viable cells were purified by LSM centrifu~ation and cultured in complete
medium supplemented with 5 units recombinant human IL-2/ml and 5 units
recombinant human IL-4/ml for up to three weeks until the cells no longer responded
to lymphokines and were considered "rested". The ability of the T cells to
2s proliferate to peptides Cry j IIA and Cry j IIB, recombinant Cry j II (rCry j II),
purified native C~y j II, or purified native C1y j I was then assessed. For assay, 2 X
104 rested cells were restimulated in the presence of 2 X 104 autologous Epstein-
Barr virus (EBV)-transformed B cells (prepared as described below) (gamma-
irradiated with 25,000 RADS) with 2-50 ~lg/ml of rCry j II, purified native C~y j
II, peptides Cry j IIA and Cry j IIB, of purified native Cry j I, in a volume of 200 ~l
complete medium in duplicate or triplicate wells in 96-well round bottom plates for
2-4 days. The optimal incubation was found to be 3 days. Each well then received .
1 ~lCi tritiated thymidine for 16-20 hours. The counts incorporated were collected
onto glass ~lber filter mats and processed for liquid scintillation counting. The
3s maximum response in a titration of each peptide is expressed as the stimulation index
(S.I.). The S.I. is the counts per minute (CPM) incorporated by cells in response to
peptide, divided by the CPM incorporated by cells in medium only. An S.I. value
~. ~ ~",. . . .. . .. .
`WO 94/1 1512 2 1 4 8 ~ 1 3 Pcr/US93/ 1 loOO ~
39 i:
equal to or greater than 2 times the background level is considered "positive" and
indicates that the peptide contains a T cell epitope. The results of this assay
indicated that peptides Cr j II, and Cry j IIB did noit appear to contain a T cell
;epitope for this particular allergenic patient. However, additional Japanese cedar
S ~ pollen allergic patients will be tested in this assay system and one or both of these
peptides may contain T cell epilopes for other allergic individuals
; ~
~.":.:
~, ;:
r > ~
.~,
~',, ~;;
.,
W094/11512 21~871:~ pCI/US93/11000 ~
Preparation of (EBV)-transformed B Cells for Use as Antigen Presenting Cells
Autologous EBV-transformed cell lines were y-irradiated with2S,000 Rad ¦
and used as antigen presenting cells in secondary proliferation assays and secondary '
bulk stimu!ations. These cell lines were also used as a control in the immuno-
s fluorescence flow cytometry analysis. These EBV-transformed cell lines were made
by incubating S X 106 PBL with 1 ml of B-59/8 Marmoset cell line (ATCC
CRL1612, American Type Culture Collection, Rockville, MD) conditioned medium
;in the presence of 1 llg/ml phorbol 12-myristate 13-acetate (PMA) at 37C for 60
minutes in 12 X 75 mm polypropylene round-bottom Falcon snap cap tubes (Becton
o ; Dickinson Labware, Lincoln Park, NJ) . These cells were then diluted to 1 .2S X 106
cellsiml in RPMI~ 0 as described above except supplemented with 10% heat-
irlactivated fetal bovine serum and cultured in 200 !11 aliquots in flat bottom culture
plates until visible colonies were detected~ They were then transferred to larger
wells until the cell !ines were established.
15~ Although the invention has been described with reference to its preferred
embodiments, othèr embodiments, can achieve the same results. Variations and
modifications to the present invention will be obvious to those skilled in the art and it
is inended to cover in the appended claims all such modification and equivalents and
fo!low in the true spirit and scope of this invention.
,~, .
.. ~ ~ . .
t
i,, `
`~:
,
, ,, . . - ` . ~ ` : : . :.
214~7~3 l~
/ c ,,.. ; ;`
WO 94/1 1512 PCI /US93/1 1000
SEQUENCE LISTING
i
(
(1) GENERAL INFORMATION:
S
(i) APPLICANT:
(A) NAME: IMMULOGIC PHARMACEUTICAL CORPORATION
(B) STREET: 610 Lincoln Street
(C) CITY: Waltham
~D) STATE: MA
(E) COUNTRY: USA
(F) POSTAL CODE (ZIP): 02154
(G) TELEPHONE: (617) 466-6000
(H) TELEFAX: (617)466-6040
(ii) TITLE OF INVENTION: Allergenic Proteins and Peptides From
Japanese Cedar Pollen
(iii) NUMBER OF SEQUENCES: 55
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: ASCII (TEXT)
`: :
:~ (v) CURRENT APPLICATION DATA:
(A) APPLIC~TION NUMBER:
: (B) FILING DATE:
~vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT. INFORMATION:
(A)- NAME: Vanstone, Darlene
(B) REGISTRATION NUMBER: 35,729
(C) REFERENCE/DOCKET NUMBER: IPC-033PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 466-6000
(B) TELEFAX: (617) 466-6040
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1726 base pairs i`
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single ~;
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA `-
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 42..1586
WO94/11512 PCI/US93/11000 ~ ~ ~14~713 42
(xi) SEQ~NCE DESCRIPTION: SEQ ID NO:l:
TGAGTTCGAG ACAAGTATAG AAAGAATTTT CTTTTATTAA A ATG GCC ATG AAA
53
Met Ala Met Lys
) `
TTA ATT GCT CCA ATG GCC TTT CTG GCC ATG CAA TTG ATT ATA ATG GCG
101
Leu Ile Ala Pro Met Ala Phe Leu Ala Met Gln Leu Ile Ile Met Ala
5 10 15 20
GCA GCA GAA GAT CAA TCT GCC CAA ATT ATG TTG GAC AGT GTT GTC GAA
149
~: Ala Ala Glu Asp Gln Ser Ala Gln Ile Met Leu Asp Ser Val Val Glu
: 25 30 35
.
AAA TAT CTT AGA TCG AAT CGG AGT TTA AGA AAA GTT GAG CAT TCT CGT
: 197
:: Lys Tyr Leu Arg Ser Asn Arg Ser Leu Arg Lys Val Glu His Ser Arg
CAT GAT GCT ATC AAC ATC TTC AAT GTG GAA AAG TAT GGC GCA GTA GGC
- 245
His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala Val Gly
: ~ 55 60 65
GAT GGA AAG CAT GAT TGC ACT GAG GCA TTT TCA ACA GCA TGG CAA GCT
: 293
Asp Gly Lys ~is Asp Cys Thr Glu Ala Phe Ser Thr Ala Trp Gln Ala
70 75 80
~ .
: 35 GCA TGC AAA AAC CCA TCA GCA ATG TTG CTT GTG CCA GGC AGC AAG AAA
341
Ala Cys Lys Asn Pro Ser Ala Met Leu Leu Val Pro Gly Ser Lys Lys
90 95 100
: TTT GTT GTA AAC AAT CTG TTC TTC AAT GGG CCA TGT CAA CCT CAC TTT
389
Phe Val Val Asn Asn Leu Phe Phe Asn Gly Pro Cys Gln Pro His Phe
105 110 115
ACT TTT AAG GTA GAT GGG ATA ATA GCT GCG TAC CAA AAT CCA GCG AGC
437
: Thr Phe Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln Asn Pro Ala Ser
120 125 130
TGG AAG AAT AAT AGA ATA TGG TTG CAG TTT GCT AAA CTT ACA GGT TTT i ;.
:~ 485
Trp Lys Asn Asn Arg Ile Trp Leu Gln Phe Ala Lys Leu Thr Gly Phe
, 135 140 145 `~
ACT CTA ATG GGT AAA GGT GTA ATT GAT GGG CAA GGA AAA CAA TGG TGG ',
533 . t:~
Thr Leu Met Gly Lys Gly Val Ile Asp Gly Gln Gly Lys Gln Trp Trp ~-
150 155 160 . j.
:: 60 ~;
: GCT GGC CAA TGT AAA TGG GTC AAT GGA CGA GAA ATT TGC AAC GAT CGT
581
Ala Gly Gln Cys Lys Trp Val Asn Gly Arg Glu Ile Cys Asn Asp Arg
165 170 175 180 .
~-: 65
GAT AGA CCA ACA GCC ATT AAA TTC GAT TTT TCC ACG GGT CTG ATA ATC
, .
':
;
-:
. ,~' WO 94/1 1512 2 1 ~ 8 7 1 3 PCI`/US93/1 1000 r~
~. -
43 ~ :
629
Asp Arg Pro Thr Ala Ile Lys Phe Asp Phe Ser Thr Gly I.eu Ile Ile
18S 190 1~5
CAA GGA CTG AAA CTA ATG AAC AGT CCC GAA TTT CAT TTA GTT TTT GGG
677
Gln Gly Leu Lys Leu Met Asn Ser Pro Glu Phe Hls Leu Val Phe Gly
200 205 210
AAT TGT GAG GGA GTA AAA ATC ATC GGC ATT AGT ATT ACG GCA CCG AGA
72S
Asn Cys Glu Gly Val Lys Ile Ile Gly Ile Ser Ile Thr Ala Pro Arg
21S 220 225
GAC AGT CCT AAC ACT GAT GGA ATT GAT ATC TTT GCA TCT AAA AAC TTT
773
Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Ala Ser Lys Asn Phe
230 235 240
CAC TTA CAA AAG AAC ACG ATA GGA ACA GGG GAT GAC TGC GTC GCT ATA
821
His Leu Gln Lys Asn Thr Ile Gly Thr Gly Asp Asp Cys Val Ala Ile
245 250 255 260
GGC ACA GGG TCT TCT AAT ATT GTG ATT GAG GAT CTG ATT TGC GGT CCA
869
Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu Ile Cys Gly Pro
265 270 275
GGC CAT GGA ATA AGT ATA GGA AGT CTT GGG AGG GAA AAC TCT AGA GCA
917
Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Glu Asn Ser Arg Ala
280 285 290
GAG GTT TCA TAC GTG CAC GTA AAT GGG GCT AAA TTC ATA GAC ACA CAA
965
Glu Val Ser Tyr Val His Val Asn Gly Ala Lys Phe Ile Asp Thr Gln
295 300 305
AAT GGA TTA AGA ATC AAA ACA TGG CAG GGT GGT TCA GGC ATG GCA AGC
1013
Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser Gly Met Ala Ser
310 315 320
CAT ATA ATT TAT GAG AAT GTT GAA ATG ATA AAT TCG GAG AAC CCC ATA
1061
His Ile Ile Tyr Glu Asn Val Glu Met Ile Asn Ser Glu Asn Pro Ile
325 330 335 340
TTA ATA AAT CAA TTC TAC TGC ACT TCA GCT TCT GCT TGC CAA AAC CAG
1109
Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ser Ala Cys Gln Asn Gln
345 350 355
AGG TCT GCG GTT CAA ATC CAA GAT GTG ACA TAC AAG AAC ATA CGT GGG
1157
Arg Ser Ala Val Gln Ile Gln Asp Val Thr Tyr Lys Asn Ile Arg Gly ~:
360 365 370 ~-
ACA TCA GCA ACA GCA GCA GCA ATT CAA CTT AAG TGC AGT GAC AGT ATG
1205
Thr Ser Ala Thr Ala Ala Ala Ile Gln Leu Lys Cys Ser Asp Ser Met
375 380 385
CCC TGC AAA GAT ATA AAG CTA AGT GAT ATA TCT TTG AAG CTT ACC TCA
1253
WO 94/ 1 1 5 1 2 ,~ 8 ~1 3 PCl / US93/ 11 000
44
. :-
Pro Cys Lys Asp Ile Lys Leu Ser Asp Ile Ser Leu Lys Leu Thr Ser
390 395 400
GGG AAA ATT GCT TCC TGC CTT AAT GAT AAT GCA AAT GGA TAT TTC AGT
1301
Gly Lys Ile Ala Ser Cys Leu Asn Asp Asn Ala Asn Gly Tyr Phe Ser
495 ~10 415 4~0
GGA CAC GTC ATC CCT GCA TGC AAG AAT TTA AGT CCA AGT GCT AAG CGA ¦.
1349
Giy His Val Ile Pro Ala Cys Lys Asn Leu Ser Pro Ser Ala Lys Arg
425 430 435
AAA GAA TCT AAA TCC CAT AAA CAC CCA AAA ACT GTA ATG GTT GAA AAT
1397
Lys Glu Ser Lys Ser His Lys His Pro Lys Thr Val Met Val Glu Asn
440 445 450
ATG CGA GCA TAT GAC AAG GGT AAC AGA ACA CGC ATA TTG TTG GGG TCG
1445
Met Arg Ala Tyr Asp Lys Gly Asn Arg Thr Arg Ile Leu Leu Gly Ser
455 460 455
AGG CCT CCG AAT TGT ACA AAC AAA TGT CAT GGT TGC AGT CCA TGT AAG
1493
Arg Pro Pro Asn Cys Thr Asn Lys Cys His Gly Cys Ser Pro Cys Lys
470 475 480
GCC AAG TTA GTT ATT GTT CAT CGT ATT ATG CCG CAG GAG TAT TAT CCT
1541
: Ala Lys Leu Val Ile Val His Arg Ile Met Pro Gln Glu Tyr Tyr Pro
485 490 495 500 ~:
CAG AGG TGG ATA TGC AGC TGT CAT GGC AAA ATC TAC CAT CCA TAATGAGATA
1593
Gln Arg Trp Ile Cys Ser Cys His Gly Lys Ile Tyr Hls Pro
505 510
CATTGAAACT GTATGTGCTA GTGAATATTC TTGTGGTACA ATATTAGAAC TGATATTGAA
1653
AATAAATCAT CAATGTTTCT AAGGCATTTA TAATAGATTA TATTAATGGT TCAGCCTGGT
1713
GCAAAAAAAA AAA
1726
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 514 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Met Lys Leu Ile Ala Pro Met Ala Phe Leu Ala Met Gln Leu
1 5 10 15
Ile Ile Met Ala Ala Ala Glu Asp Gln Ser Ala Gln Ile Met Leu Asp
~` W094/11512 214S713 PCI/US93/11000
4 5 s
Ser Val Val Glu Lys Tyr Leu Arg Ser Asn Arg Ser Leu Arg Lys Val
Glu His Ser Arg His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr ¦
50 55 60
Gly Ala Val Gly Asp Gly Lys His Asp Cys Thr Glu Ala Phe Ser Thr
65 70 75 80
Ala Trp Gln Ala Ala Cys Lys Asn Pro Ser Ala Met Leu Leu Val Pro
9Q 95
Gly Ser Lys Lys Phe Val Val Asn Asn Leu Phe Phe Asn Gly Pro Cys
100 105 110
Gln Pro His Phe Thr Phe Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln
115 120 125
Asn Pro Ala Ser Trp Lys Asn Asn Arg Ile Trp Leu Gln Phe Ala Lys
130 135 140
. ~ Leu Thr Gly Phe Thr Leu Met Gly Lys Gly Val Ile Asp Gly Gln Gly
145 150 155 1~0
Lys Gln Trp Trp Ala Gly Gln Cys Lys Trp Val Asn Gly Arg Glu Ile
165 170 175
Cys Asn Asp Arg Asp Arg Pro Thr Ala Ile Lys Phe Asp Phe Ser Thr
180 185 190
~:~ Gly Leu Ile Ile Gln Gly Leu Lys Leu Met Asn Ser Pro Glu Phe His
: 195 200 205
' ~";
.~
Leu Val Phe Gly Asn Cys Glu Gly Val Lys Ile Ile Gly Ile Ser Ile
210 215 220
:: Thr Ala Pro Arg Asp Ser Pro Asn Thr Asp Gly Ile Asp Ile Phe Ala
225 230 235 240
Ser Lys Asn Phe His Leu Gln Lys Asn Thr Ile Gly Thr Gly Asp Asp
245 250 255
Cys Val Ala Ile Gly Thr Gly Ser Ser Asn Ile Val Ile Glu Asp Leu
260 265 270
Ile Cys Gly Pro Gly His Gly Ile Ser Ile Gly Ser Leu Gly Arg Glu
275 280 285
Asn Ser Arg Ala Glu Val Ser Tyr Val His Val Asn Gly Ala Lys Phe
2~0 295 300
Ile Asp Thr Gln Asn Gly Leu Arg Ile Lys Thr Trp Gln Gly Gly Ser
305 310 315 320 ~.
Gly Met Ala Ser His Ile Ile Tyr Glu Asn Val Glu Met Ile Asn Ser
325 330 335 P:~
Glu Asn Pro Ile Leu Ile Asn Gln Phe Tyr Cys Thr Ser Ala Ser Ala
340 345 350
Cys Gln Asn Gln Arg Ser Ala Val Gln Ile Gln Asp Val Thr Tyr Lys
355 360 365
Asn Ile Arg Gly Thr Ser Ala Thr Ala Ala Ala Ile Gln Leu Lys Cys
370 375 380
WO94/11512 2 1 ~87 13 PCT/US93/11000 ~
46 ~:
Ser Asp Ser Met Pro Cys Lys Asp Ile Lys Leu Ser Asp Ile Ser Leu
385 390 395 400
Lys Leu Thr Ser Gly Lys Ile AIa Ser Cys Leu Asn Asp Asn Ala Asn
405 410 415 ~ I
Gly Tyr Phe Ser Gly His Val Ile Pro Ala Cys Lys Asn Leu Ser Pro 4
420 425 430
, ,
Ser Ala Lys Arg Lys Glu Ser Lys Ser His ky- His Pro Lys Thr Val
435 440 44s
Met Val Glu Asn Met Arg Ala Tyr Asp Lys Gly Asn Arg Thr Arg Ile ~ .
450 455 460 :~.
: Leu Leu Gly Ser Arg Pro Pro Asn Cys Thr Asn Lys Cys His Gly Cys
465 470 475 480
20:
: ~ Ser Pro Cys Lys Ala Lys Leu Val Ile Val His Arg Ile Met Pro Gln
485 490 49S
Glu Tyr Tyr Pro Gln Arg Trp Ile Cys Ser Cys His Gly Lys Ile Tyr
s00 505 S10
:
His Pro
,
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
:::
40~ (v) FRAGMENT TYPE: internal
: ' ' ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Arg Lys Val Glu His Ser Arg His Asp Ala Ile Asn Ile Phe Asn Val
1 5 10 15
Glu Lys Tyr Gly Ala Val Gly Asp Gly Lys His Asp Cys Thr Glu Ala
Phe Ser Thr Ala Trp Gln Ala Ala Cys Lys Asn Pro Ser
35 : 40 45
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE OEARACTERISTICS: ;.
(A) LENGTH: 41 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
: ~v) FRAGMENT TYPE: internal
:
'` W094/llSi2 2148713 PCI/U593/11000
4 7 ~i .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Arg Lys Val Glu His Ser Arg His Asp Ala Ile Asn Ile Phe Asn Val .
1 5 10 15
Glu Lys Tyr Gly Ala Val Gly Asp Gly Lys His Asp Cys Thr Glu Ala
20 25 30
Phe Ser Thr Ala Trp Gln Lys Asn Pro
35 40
~2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE C~ARACTERISTICS:
~A) LENGTH: 36 amino acids
(B~ TYPE: amino acid
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ser Arg His Asp Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr Gly Ala
1 5 10 15
Val Gly Asp Gly Lys His Asp Cys Thr Glu Ala Phe Ser Thr Ala Trp
20 ~ 25 30
Gln Lys Asn Pro
(2) INFORMATION FOR SEQ ID NO:6:
(i) 5EQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ala Ile Asn Ile Phe Asn Val Glu Lys Tyr
1 5 10 ~-
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1410 base pairs '.-
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
WO94/1151~ 713 PCI/US93/11000 '~ ~;
48
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: '~
AGAAAAGTTG AGCATTCTCG TCATGATGCT ATCAACATCT TCAATGTGGA AAAGTATGGC ,
.
GCAGTAGGCG ATGGAAAGCA TGATTGCACT GAGGCATTTT CAACAGCATG GCAAGCTGCA~
120
TGCAAAAACC CATCAGCAAT GTTGCTTGTG CCAGGCAGCA AGAAATTTGT TGTAAACAAT
180
CTGTTCTTCA ATGGGCCATG TCAACCTCAC TTTACTTTTA AGGTAGATGG GATAATAGCT
240
GCGTACCAAA ATCCAGCGAG CTGGAAGAAT AATAGAATAT GGTTGCAGTT TGCTAAACTT
300
ACAGGTTTTA CTCTAATGGG TAAAGGTGTA ATTGATGGGC AAGGAAAACA ATGGTGGGCT
360
GGCCAATGTA AATGGGTCAA TGGACGAGAA ATTTGCAACG ATCGTGATAG ACCAACAGCC
420
ATTAAATTCG ATTTTTCCAC GGGTCTGATA ATCCAAGGAC TGAAACTAAT GAACAGTCCC
480
GAATTTCATT TAGTTTTTGG GAATTGTGAG GGAGTAAAAA TCATCGGCAT TAGTATTACG
540
GCACCGAGAG ACAGTCCTAA CACTGATGGA ATTGATATCT TTGCATCTAA AAACTTTCAC
600
TTACAAAAGA ACACGATAGG AACAGGGGAT GACTGCGTCG CTATAGGCAC AGGGTCTTCT
660
AATATTGTGA TTGAGGATCT GATTTGCGGT CCAGGCCATG GAATAAGTAT AGGAAGTCTT
720
GGGAGGGAAA ACTCTAGAGC AGAGGTTTCA TACGTGCACG TAAATGGGGC TAAATTCATA
780
GACACACAAA ATGGATTAAG AATCAAAACA TGGCAGGGTG GTTCAGGCAT GGCAAGCCAT
840
ATAATTTATG AGA~TGTTGA AATGATAAAT TCGGAGAACC CCATATTAAT AAATCAATTC
900
TACTGCACTT CAGCTTCTGC TTGCCAAAAC CAGAGGTCTG CGGTTCAAAT CCAAGATGTG
960
ACATACAAGA ACATACGTGG GACATCAGCA ACAGCAGCAG CAATTCAACT TAAGTGCAGT
1020
GACAGTATGC CCTGCAAAGA TATAAAGCTA AGTGATATAT CTTTGAAGCT TACCTCAGGG
1080
AAAATTGCTT CCTGCCTTAA TGATAATGCA AATGGATATT TCAGTGGACA CGTCATCCCT ;`
1140
GCATGCAAGA ATTTAAGTCC AAGTGCTAAG CGAAAAGAAT CTAAATCCCA TAAACACCCA
1200
AAAACTGTAA TGGTTGAAAA TATGCGAGCA TATGACAAGG GTAACAGAAC ACGCATATTG
1260
TTGGGGTCGA GGCCTCCGAA TTGTACAAAC AAATGTCATG GTTGCAGTCC ATGTAAGGCC
¦ r ~
~;`` W O 94/llS12 214S7 1 ~ PCT/US93tll000
49
1320
AAGTTAGTTA TTGTTCATCG TATTATGCCG CAGGAGTATT ATCCTCAGAG GTGGATATGC
1380
AGCTGTCATG GCAAAATCTA CCATCCATAA ~ 3
1410
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 1395 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
~5 TCTCGTCATG ATGCTATCAA CATCTTCAAT GTGGAAAAGT ATGGCGCAGT AGGCGATGGA
AAGCATGATT GCACTGAGGC ATTTTCAACA GCATGGCAAG CTGCATGCAA AAACCCATCA
120
GCAATGTTGC TTGTGCCAGG CAGCAAGAAA TTTGTTGTAA ACAATCTGTT CTTCAATGGG
180
CCATGTCAAC CTCACTTTAC TTTTAAGGTA GATGGGATAA TAGCTGCGTA CCAAAATCCA
240
GCGAGCTGGA AGAATAATAG AATATGGTTG CAGTTTGCTA AACTTACAGG TTTTACTCTA
300
ATGGGTAAAG GTGTAATTGA TGGGCAAGGA AAACAATGGT GGGCTGGCCA ATGTAAATGG
360
GTCAATGGAC GAGAAATTTG CAACGATCGT GATAGACCAA CAGCCATTAA ATTCGATTTT
420
TCCACGGGTC TGATAATCCA AGGACTGAAA CTAATGAACA GTCCCGAATT TCATTTAGTT :
480
TTTGGGAATT GTGAGGGAGT AAAAATCATC GGCATTAGTA TTACGGCACC GAGAGACAGT
540
CCTAACACTG ATGGAATTGA TATCTTTGCA TCTAAAAACT TTCACTTACA AAAGAACACG ;~
600
ATAGGAACAG GGGATGACTG CGTCGCTATA GGCACAGGGT CTTCTAATAT TGTGATTGAG
-660
GATCTGATTT GCGGTCCAGG CCATGGAATA AGTATAGGAA GTCTTGGGAG GGAAAACTCT
720
AGAGCAGAGG TTTCATACGT GCACGTAAAT GGGGCTAAAT TCATAGACAC ACAAAATGGA
780
TTAAGAATCA AAACATGGCA GGGTGGTTCA GGCATGGCAA GCCATATAAT TTATGAGAAT
840
W0 94/11512 i~ 1 ~ 8 ~1 3 PCr/US93/11000
~ :
GTTGAAATGA TAAATTCGGA GAACCCCATA TTAATAAATC AATTCTACTG CACTTCAGCT
900
TCTGCTTGCC AAAACCAGAG GTCTGCGGTT CAAATCCAAG ATGTGACATA CAAGAACATA ~ :~
960
CGTGGGACAT CAGCAACAGC AGCAGCAATT CAACTTAAGT GCAGTGACAG TATGCCCTGC
1020
AAAGATATAA AGCTAAGTGA TATATCTTTG AAGCTTACCT CAGGGAAAAT TGCTTCCTGC
1080
CTTAATGATA ATGCAAATGG ATATTTCAGT GGACACGTCA TCCCTGCATG CAAGAATTTA
1140
AGTCCAAGTG CTAAGCGAAA AGAATCTAAA TCCCATAAAC ACCCAAAAAC TGTAATGGTT
1200
GAAAATATGC GAGCATATGA CAAGGGTAAC AGAACACGCA TATTGTTGGG GTCGAGGCCT
1260
CCGAATTGTA CAAACAAATG TCATGGTTGC AGTCCATGTA AGGCCAAGTT AGTTATTGTT
1320
25 - CATCGTATTA TGCCGCAGGA GTATTATCCT CAGAGGTGGA TATGCAGCTG TCATGGCAAA
1380
ATCTACCATC CATAA
1395
.
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1479 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPO~OGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: :;
GAAGATCAAT CTGCCCAAAT TATGTTGGAC AGTGTTGTCG AAAAATATCT TAGATCGAAT
CGGAGTTTAA GAAAAGTTGA GCATTCTCGT CATGATGCTA TCAACATCTT CAATGTGGAA
120
AAGTATGGCG CAGTAGGCGA TGGAAAGCAT GATTGCACTG AGGCATTTTC AACAGCATGG `~
180
CAAGCTGCAT GCAAAAACCC ATCAGCAATG TTGCTTGTGC CAGGCAGCAA GAAATTTGTT
240
.GTAAACAATC TGTTCTTCAA TGGGCCATGT CAACCTCACT TTACTTTTAA GGTAGATGGG
300
ATAATAGCTG CGTACCAAAA TCCAGCGAGC TGGAAGAATA ATAGAATATG GTTGCAGTTT
360
GCTAAACTTA CAGGTTTTAC TCTAATGGGT AAAGGTGTAA TTGATGGGCA AGGAAAACAA : :
420
.... WO94/11512 214~71 3 PCT/US93/11000
TGGTGGGCTG GCCAATGTAA ATGGGTCAAT GGACGAGAAA TTTGCAACGA TCGTGATAGA
48Q
CCAACAGCCA TTAAATTCGA TTTTTCCACG GGTCTGATAA TCCAAGGACT GAAACTAATG
540
AACAGTCCCG AATTTCATTT AGTTTTTGGG AATTGTGAGG GAGTAAA~AT CATCGGCATT ' `
600
AGTATTACGG CACCGAGAGA CAGTCCTAAC ACTGATGGAA TTGATATCTT TGCATCTAAA
660
AACTTTCACT TACAAAAGAA CACGATAGGA ACAGGGGATG ACTGCGTCGC TATAGGCACA
720
GGGTCTTCTA ATATTGTGAT TGAGGATCTG ATTTGCGGTC CAGGCCATGG AATAAGTATA
780
GGAAGTCTTG GGAGGGAAAA CTCTAGAGCA GAGGTTTCAT ACGTGCACGT AAATGGGGCT
~0. 840
` AAATTCATAG ACACACAAAA TGGATTAAGA ATCAAAACAT GGCAGGGTGG TTCAGGCATG
- 900
GCAAGCCATA TAATTTATGA GAATGTTGAA ATGATAAATT CGGAGAACCC CATATTAATA ~.
960
AATCAATTCT ACTGCACTTC AGCTTCTGCT TGCCAAAACC AGAGGTCTGC GGTTCAAATC
1020
3o
CAAGATGTGA CATACAAGAA CATACGTGGG ACATCAGCAA CAGCAGCAGC AATTCAACTT
1080
AAGTGCAGTG ACAGTATGCC CTGCAAAGAT ATAAAGCTAA GTGATATATC TTTGAAGCTT ~.
~35 1140 .
ACCTCAGGGA AAATTGCTTC CTGCCTTAAT GATAATGCAA ATGGATATTT CAGTGGACAC ~
1200 .
GTCATCCCTG CATGCAAGAA TTTAAGTCCA AGTGCTAAGC GAAAAGAATC TAAATCCCAT
1260
AAACACCCAA AAACTGTAAT GGTTGAAAAT ATGCGAGCAT~ATGACAAGGG TAACAGAACA
1320 ` ::
::
CGCATATTGT TGGGGTCGAG GCCTCCGAAT TGTACAAACA AATGTCATGG TTGCAGTCCA
1380
TGTAAGGCCA AGTTAGTTAT TGTTCATCGT ATTATGCCGC AGGAGTATTA TCCTCAGAGG
1440
TGGATATGCA GCTGTCATGG CAAAATCTAC CATCCATAA
1479
~ `
~2) INFORMATION FOR SEQ ID NO:10:
~i) SEQUENCE CHARACTERISTICS: .
~A) LENGTH: 35 base pairs
~B) TYPE: nucleic acid ~-
~C) STRANDEDNESS: single `~ .
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
`
W094/11512 2,~48rl 13 ~ PCr/US93/11000 ~- ~
52
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGGTCTAGAG GTACCGTCCG TCCGATCGAT CCATT
S
(2~ INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) ST ~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AATGATCGAT GCT
13
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
RTAYTTYTCN ACRTTRAA
18
~2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
5~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GGGTCTAGAG GTA
13
~ `
(2) INFORMATION FOR SEQ ID NO:14:
:
: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
~ W 0 94/11512 ~14~713 PCT/US93/llOOO
53 ~;
(v) FRAGMENT TYPE: internal
~xi) SEQUENCE DESCRIPTION: SEQ ID NO :14:
Phe Asn Val Glu Lys Tyr
1 5
- ~10
(2) INFORMATION FOR SEQ ID NO:15:
~ i) SEQUENCE CHARACTERISTICS:
s-~ (A) LENGTH; 27 base pairs
~15 ~ TYPE: nucleic acid
~ (C) STRANDEDNESS: single
`~: (D) TOPOLOGY: linear
ii) MOLECULE TYPE: cDNA
~20 : ~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
CCTGCAGTAY TTYTCNACRT TRAANAT
27
(2) ~INFORMATION FOR SEQ ID NO:16:
,30~ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 base pairs
: (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
. ~
~40 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCTGCAG
:~`45
~ ~:
:: (2) INFORMATION FOR SEQ ID NO:17:
~ ~i) SEQUENCE CHARAcTERIsTIcs
:~SO (A) LENGTH: 7 amino acids .
. (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
` (v) FRAGMENT TYPE: internal :~-
:- ; ~ :.
~60 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Ile Phe Asn Val Glu Lys Tyr 5
1 5
~ ` ~
(2) INFORMATION FOR SEQ ID NO:18:
~`,
~ ' ;
.~ , .
W 0 94/1l512 pCT/US93/11000 .~
214~7~3 54
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH. 27 base pairs I -
(B) TYPE: nucleic acid l:~
(C) STRANDEDNESS: single `:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CCTGCAGTAY TTYTCNACRT TRAADAT
5 27
(2) INFORMATION FOR SEQ ID NO:l9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MO~ECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GCNATHAAYA THTTYAA
: 17
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amlno acid
(D) TOPOLOGY: linear '
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Ala Ile Asn Ile Phe Asn
1 5
~2) INFORMATION FOR SEQ ID NO:21:
(i) SEQOE NCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
WO 94/11512 2 1~ ~ 71 3 PCr/US93/11000 i`i
GGAATTCCGC NATHAAYATH TTYAAYGT
~2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs ?
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GGAATTCC
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
¦l ~ (D) TOPOLOGY: linear
3 (ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Ala Ile Asn Ile Phe Asn Val
1 5
(2) INFORMATION FOR SEQ ID NO:24:
~ (i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
. (C) STRANDEDNESS: single
(D) TOPOLOGY linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
GCYTCNGTRC ARTCRTGYTT
3 20 ~i
`3. ( 2) INFORMATION FOR SEQ ID NO:25: --
1 60 (i) SEQUENCE CHARACTERISTICS: ~
' (A) LENGTH: 7 amino acids ~.
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
,
.,
Wo94/11512 21 ~713 PCr/US9~/11000 1`
56
(v) FRAGMENT TYPE: internal
S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Lys His Asp Cys Thr Glu Ala
l 5
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: llnear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
~5
GGCTGCAGGT RCARTCRTGY TTNCCRTC
28
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
~ (C) STRANDEDNESS: single
¦ 35 (D) TOPOLOGY: linear
¦ (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
GGCTGCAG
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide `
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Asp Gly Lys ~is Asp Cys Thr .
1 5
(2) INFORMATION FOR SEQ ID NO:29:
~W094/11512 2la~ 3 PCI/U593/1100
I
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid .
S (C) STRANDEDNESS: single
(D~ TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
1:0
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
ATGTTGGACA GTGTTGTCGA A
21
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
: (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
,: :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
~30: ~ :
GGGAATTCAG AA~AGTTGAG CATTCTCGT
29
~ (2) INFORMATION FOR SEQ ID NO:31:
j 35
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~:40 (D) TOPOLOGY: linear
~- (ii) MOLECULE TYPE: cDNA
~'-;
(xi) SEQOE NCE DESCRIPTION: SEQ ID NO:31:
GGGAATTC
.
,
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs .
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~: (ii) MOLECULE TYPE: cDNA
'
. 65
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
W 0 94/11512 PC'r/US93/11000 ~
2143713 58
~ GTTCTTCAAT GGGCCATGT .
19
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS: ;~
(A) LENGTH: 20 base pairs
- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
: (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xl) SEQUENCE DESCRIPTION: SEQ ID NO:33:
GTGTTAGGAC TGTCTCTCGG
~2) INFORMATION FOR SEQ ID NO:34:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
:: (D) TOPOLOGY: linear
~: 30 (ii) MOLECULE TYPE: cDNA
~ .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
TGTCCAGGCC ATGGAATAAG
:
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 base pairs
, (B) TYPE: nucleic acid
. (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ 50 (ii) MOLECULE TYPE: cDNA
.~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
S5 i.
GCCTTACATG GACTGCAACC
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
-~ (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
~ 65 (D) TOPOLOGY: linear
J.~
~'
~ ~ WOg4/11512 21~S~l~ Pcr/us93/llooo ~
59
(ii) MOLECULE TYPE cDNA
:'
5~ (xi) SEQUENCE DESCRIPTION SEQ ID NO 36 _
TCCACGGGTC TGATAATCCA
2~INFORMATION FOR SEQ ID NO 37
SEQUENCE CHARACTERISTICS
(A) LENGTH 20 base pairs
(B)~TYPE nucleic acid
15~ (C) STRANDEDNESS single
(D~TOPOLOGY linear
~ ( i i ? MOLECULE TYPE cDNA
(x~)~SEQOE NC~ DECCRIPTIO~ SEQ ID NO 37
; AGGCAGGAAG CAATTTTCCC
~25~ 20 ~
21~NF M~TIGN FOR 53Q~ID NO~38
`(i3 SEQUENCE CHARACTERISTICS
(A) LENGTH `20 base pairs
(B) TYPE ~nucleic acid
(C) STRANDE NESS single
~5 ~ (D) TOPOLOGY~ linear
ii) MOLECULE TYPE cDNA
(xi) SEQ OENCE DESCRIPTION SEQ ID NO 38
TACTGCACTT CAGCTTCTGC
f -''" , ~ ~ 20
,-~45
2~INFORMATION FOR SEQ ID NO 39
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 base pairs
~50 (B) TYPE nuclelc acid }
(C) STRANDEDNESS single s
~ (D) TOPOLOGY linear
;~ (ii) MOLECULE TYPE cDNA
~55 ~ ;`
(xi) SEQUENCE DESCRIPTION SEQ ID NO 39
~`-60 GGGGGTCTCC GAATTTATCA
(2) ~INFORMATION FOR SEQ ID NO 40
~6S~ (i) SEQUENCE CHARACTERISTICS
(A) LENGTH 20 base pairs
1` ~'-`
2 1 4 .3 7 1 3
WO 94/ 1 1 5 1 2 PCr/ ~IS93/ 1 1 000 1 . .
i~`
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: CDNA . -
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GGATATTTCA GTGGACACGT
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2G base pairs
~B) TYPE: nucleic acid
: (C) STRANDEDNESS: single
(D) TOPOLOGY~ linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
. ~
¦ ~ 30 TATTAGAAGA CCCTGCGCCT
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
¦ (C) STRANDEDNESS: single
I (D) TOPOLOGY: linear
140
¦ (ii) MOLECULE TYPE: cDNA
1` .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
CCATGTAAGG CCAAGTTAGT
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single .
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
1,
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
: - ACACCTTTAC CCATTAGAGT
:
`~
;~ 214~713 1~
WO 94/1 1512 PCr/US93/1 1000 ~'
61 r
~: i
(2) INFORM~TION FOR SEQ ID NO:44:
~ 5 (i) SEQUENCE CHARACTERISTICS:
`:~ (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
` ~; (D) TOPOLOGY: linear
`'` ~`'10 ~
ii) MOLECULE TYPE: cDNA
, ~
~:.15~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
CTGTCCAACA TAATTTGGGC
.: ~ 20
~20 ~ :
(2) INFORMATION FOR SEQ ID NO:45:
~ (i) SEQUENCE CHARACTERISTICS:
;~ (A) LENGTH: 20 base pairs
~25 ~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
CATGGCAGGG TGGTTCAGGC
~ ~ .
(2) INFORMATION FOR SEQ ID NO:46:
~ (i) SEQUENCE CHARACTERISTICS:
G~ : ~ (A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
:: (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~45
(ii) MOLECULE TYPE: cDNA
-50 ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
TAGCCCCATT TACGTGCACG
55~
~2) INFORMATION FOR SEQ ID NO:47: ` ~.
SEQUENCE CHARACTERISTICS:
:~60 ~A) LENGTH: 20 base pairs
~ B) TYPE: nucleic acid ~
: ~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
,
~:65 (ii) MOLECULE TYPE: cDNA
:
~:
:
~ .
~;~: ....... .
W O 94J11512 PCTtUS93/11000 `~
4 8 7 1 3 62 11``
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
~: 5 TTGGGGTCGA GGCCTCCGAA
~ (2) INFORMATION FOR SEQ ID NO:48:
;~ 10 (i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 9 base pairs
: (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
: (D) TOPOLOGY: linear
1:5 (ii) MOLECULE TYPE: cDNA
t~ -~
.~ 20:~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
TAAAAUGGC
(2) INFORMATION FOR SEQ ID NO:49:
~: (i) SEQUENCE CHARACTERISTICS:
: (A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
;~35
~ .
~:~ (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
:
AACAAUGGC
: 40 9
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
. (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DE5CRIPTION: SEQ ID NO:50
GCCGAATTCA TGGCCATGAA ATTAATT
27
(2) INFORMATION FOR SEQ ID NO:51:
; (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
,~
2 .
WC~ 94/11512 214 8 713 PCI/US93/11000 Ir ~ `
63
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
S ~ ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5l:
GCCGAATTC t
(2) INFORMATION FOR SEQ ID NO:52:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
CGGGGATCCT CATTATGGAT GGTAGAT
27
, ~2) INFORMATION FOR SEQ ID NO:53:
3 (i) SEQUENCE CHARACTERISTICS:
'35 ~A) LENGTH: 9 base pairs
J (B) TYPE: nucleic acid
~C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~40 (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
~45
CGGGGATCC
~50
( 2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
~D) TOPOLOGY: linear ~.
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Phe Thr Phe Lys Val Asp Gly Ile Ile Ala Ala Tyr Gln
~1
t ~
WO 94111~12 21~ ~ 71 3 PCr/US93/11000 ~ ~
64 1.
1 5 10
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acld ~`
10(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGM$NT TYPE: internal
1~
(xi) SEQUENGE DESCRIPTION: SEQ ID NO:55:
Asn Gly Tyr Phe Ser Gly His Val Ile Pro Ala Cys Lys Asn
1 5 10
