Note: Descriptions are shown in the official language in which they were submitted.
~I Wo 9S112662 2 1 ~ 5 ~ 9 0 PC~IUS94/12624
MET}70D OF P~ODVCING r~7T
DIM16~IC 13N?Z YME
n~ 7. c~--n~ ~f 7-h~ Tnvf~n~ n
The analysis of creatine kinase (CK) isoforms
is important for the early diagnosis of acute myocardial
10 infarction (AMI) and for the early det~orminAtion of coronary
artery reperfusion in patients treated with thrombolytic
therapy (Alan and Wu, rlAhoratorv M~rl;c;ne, 23 (5) :297-302,
1992 ) . The CR isoforms CKI~M and CKMB can be used to
determine the success of reperfusion therapy, although
5 meabuL. t of MB isoforms provides the earliest and most
definitive results. Levels of ~ isoforms are also elevated
in patients with skeletal muscle disease, and together with
the relative MB index, can be useful for de~Prm;n;n~-S whether
the muscle damage is acute or chronic.
Pure CK~IB is needed for research studies of
myocardial metaholism and the enzyme ~ s catalytic ~ hAn; m
and for preparation of standards and ~uality control
materials for clinical analysis. Current methods of
obtaining CK~B involve homogenlzing heart tissue and
25 precipitating the CKMB with ethanol or ammonium sulfate
followed by ion exchange, gel filtration, and/or affinity
chromatography (Grace and Roberts, ~~l;n ~~h~m ~-ta, 12~:59-
71, 1982; and Herman and Roberts, ~nAl Bio~-h~m, 106:244-252,
1980) . These procedures involve multiple column-
30 purification steps, are long and tedious, and may result in
poor yields and low specific activities. In addition, the
extracts of CI~MB can contain large amounts of contAm;nAntq,
such as albumin which co-fractionates and co-migrates with
C~MB on chromatography and pathogens re~uiring special
3 5 hAn(ll ;n--,,
A need exists to improve and simplify the
production of the different isoforms of CK to satisfy the
rec,uirements of researchers and rl;ni~ 3nq
W095112662 ; PCFIUS94/12624 ~
21~rj9~ - 2 -
S v of~ th~a Invention
This ;n~r~nt;n"~ relates to a method of producing
a rP~ ' ,; nAnt eukaryotic heterodimeric enzyme in an active
form usi~g a prokaryotic host. The method involves
5 constructing a first 3NA vector containing DNA encoding one
of the subunits of the heterodimeric enzyme and, then
constructing a second DNA vector c~mt~;n;n~ DNA encoding the
second subunit of the enzyme. Once the DNA vectors are
constructed they are used to transform a prokaryotic host.
10 The transformed prokaryotic host cell is then cultured under
conditions appropriate for the~ ~xpression of the
heterodimeric enzyme. For example, using the method of the
E~resent invention, the heterodimeric ios:Eorm of creatine
kinase, i.e., C~NB, can be produced.
I 5 This invention further relates a method oi
producing a recombinant human dimeric enzyme in an active
form using a prokaryotic host.
Furthermore, the present inYention relates to
the rprnrrhi n=lnt enzyme products produced using the method of
2 0 the present invention .
The invention further relates to a transformed
prokaryotic host constructed by the method of the present
invention .
2 5 Brief Descril~tion of tllf' Drawina
Figure 1 a schematic illustration of the method
of producing a rpr~mhinr~nt dimeric enzyme of the present
invention .
3 0 I~n; ~ scr;ntion of the Inventio~
This invention is based upon the discovery that
;ffP~Gnt isoforms of creatine kinase ~i.e., C~'~BB, CK[~I and
C~MB) can be produced by constructing a DNA vector for the
two different subunits of the enzyme and tr~ncfrn~;n~ a host
35 cell with the two DNA vectors, the resulting transformed
host cell being capable of expressing CKBB, CKM~I and CKMB.
DNA v~ctor
The term "DNA vector~' is intended any
4 0 replication competent vector which has the capability of
having a DNA fragment inserted into it and, subse~uently,
the expression of that DNA insert by an appropriate host
W095/~2662 2 1 7 ~ 5 3 ~ PCFrUS94/12624
cell. In addition, the DNA vector must be receptive to t~e
insertion of a DNA fragment containing the DNA where the
sec7uence encodes the subunits of the target eukaryotic
dimeric enzyme such as creatine kinase ~i.e., Cl~M and CRB).
S E'urth. - e, the DNA vector must contain a promoter which
can be recognized by the host cell . Procedures f or the
construction of DNA vectors include those described in
MAniAtiC et al., Moler111Ar Clon;n~, A LAhr~ratrrv MAnllAl, 2d,
Cold Spring Harbor Laboratory Press (1989), herein referred
0 to as Maniatis et al..
The term ~DNA fragment" is intended to
Pnr~ ~qs any DNA fragment that encodes an enzyme subunit.
The DNA ragment once inserted into a DNA vector should be
transmittable to a host microorganism by transformation or
15 conjugation or transfection. Procedures for the
construction or extraction of DNA fragments include those
described in Maniatis et al, and others known by those
skilled in the art.
2 0 Host
The trans~ormed prokaryotic host of the present
invention can be created by various methods by those skilled
in the art. For example, transfection, trancformation or
el~,LL~ ,Lcltion as explained by Maniatis et al. can be used.
2 5 By the term ~prokaryotic ~ost" is intended any
prokaryote capable of the uptake and expression of foreign
DNA, i . e., DNA not originally a part of the prokaryotes ~ s
nuclear material. Suitable prokaryotes may include
Corynebacterium, Escherichia, Stre~tacyces or RAri 7 7~,c,
7~ t Dlm~r i c 7!:~zy~n~
The rP~ ~; nAnt dimeric enzyme of the present
invention is int~n~lP~ to rn~ _ .C~ any protein consisting of
two subunits and possessing enzymatic properties.
3 5 The invention will be further illustrated by
the fol 1 owing non-limiting 7~YPmrl i ~ication:
WO 9~112662 , PC~IUS94/12624 ~
2 1 ~
-- 4 --
~lr~MPLIFICA~ION
MatQrials and ~thods:
Clonin~ (`KMR .-r)N~ llqin~ PCR
DNA f ragments carrying cDNA encoding CKM and
5 CKB proteins that had been cloned from a human cDNA library
have been descr~bed (Perryman et al, Bio~llP~ . ~In~ l Biol~hvs .
E~P:~qP;Ircl~ C .. 140: 981-989, 1986; and Villarreal-Levy et
al ., Bi~ Pnl . ~n~l Bio~hvs . Reasearch Comm. . 144 :1116-1127,
1987~ . We used PCR ampl;fi~=t; ~n to change the DNA sequence
10 at the N- and C- termini t~ add restriction en~yme sites
that were suitable for cloning the CK cDNA into Genzyme
expression vectors . PCR primers were syntl~Pq; 7.P~l at Genzyme.
The primers had the following 5 - 3 sequences;
sL22 (Ndel site at the ATG start codon of CKB) GCC CAT ATG
CCC TTC TCC A~C AGC CAC A
SL23 (Eco~1 site after the stop codon of CKB~ GGA ATT CAT
TTC TGG GCA GGC ATG AGG
SL2~ (Ndel site at the ATG start codon of CKM) GCC CAT ATG
CCA TTC GGT AAC ACC CAC AAC
20 SL25 (Bam~1 site after the stop codon of C}~M) GCA GGA TCC
TAC TTC TGG GCG GGG ~TC AT.
The GeneAmp PCR Reagent ~it with AmpliTaq DNA
Polymerase from Perkin Elmer Cetus (Norwalk, CT) was used
for PCR reactions. The reactions were carried out following
2 5 standard procedures outlined in the literature enclosed in
the kit Specifically, 5-30 ng DNA, 100 pmol primer DNA,
2.5 U AmpliTaq DNA polvmerase, and 200 ,umol dATP, dCTP,
dGTP, dTTP were mixed with supplied buffer, and the reaction
mix was overlayed with Ampliwax ( Perkin Elmer Cetus ) . The
3 0 PCR machine (Coy Laboratory Products, Inc . Grass Lake, MI)
was ~L yL -d for ~ cycle of 94-C Lmelt) for 2 minutes,
55 C (anneal) for 2 minutes, 72 C (extend~ for 2 minutes,
this cycle was repeated 1~3 times. A final extension step
was run for 10 minutes to allow for complete polymerization
3 5 of all strands. PCR product (approximately 5-10 llg~ was
digested with Ndel and EcoR1 ~for CKB~ or Ndel and BamH1
~ for CKM) and purified by electrophoresis through a 0.7% low
melting point agarose TAE buffered gel (FMC BioProducts,
Inc. Rockland, ME~ for cloning into expression vectors.
40 Electrophoresis was performed as described in Molecular
Cloning (Sambrook, Fritsch and M~n;~ti c, 198g. Cold Spring
Harbor Press, Cold Spring Har~or, NY) . Restriction
WO 95112662 217 rj S ~l ~ PC~IUS94/12624
endonucleases were purchased from New England BioLabs
(Beverly, MA) and digestion reactions set up as suggested by
the manufacturer. DNA fragments were purified from gel
slices using the f.~nf~ An kit (BiolO1, La Jolla, CA~.
s
Conqtruct;n~ thf~ S;~n - ~rnreccinn Vector r-R7~8
The expression vector was constructed at
Genzyme as a derivative of the plasmid pBluescript SK +/-
~available from Stratagene, La Jolla, CA). Expression is
10 driven off the Lac promoter. Our vector, pRZ38, was
constructed by adding an restriction enzyme site at the ATG
start codon of the b-galactosidase gene through
site-directed mutagenesis. Mutagenesis protocols were
followed as described using the Muta-Gene In Vitro
15 Mutagenesis Rit from BioRad (Ri rl 1 , CA) . This change in
the vector allows cloning of foreign genes after the LacZ
promoter but maintains similar spacing from the promoter as
in the native gene.
20 ('lnn;n~t CKM ein~ CKR nN!~ ;nt~ R7~8
Vector DNA (5 llg) was digested with Ndel and
EcoR1 (for CKB) or Ndel and BamH1 (for CKM~ and gel purified
as above. Approximately 100 ng digested vector DNA and 100
ng digested PCR product were ligated in a 20 111 reaction (T4
25 DNA ligase purchased from NEB, Beverly, MA) . Ligation
reactions set up as described in Molecular Cloning. After
overnight ;n llh~t;nn at 15 C ligation mixes were diluted to
60~1 with dlI20. 1111 of each was electroporated into
electroporation competent E. coli strain MC1061. The
3 0 CeLl-Porator and Voltage Booster were purchased from
Bethesda Research Labs (Bethesda, MD~ . Protocols or
competent cell preparation and ele.LL~.~ v, ltion are described
in the I~struction manual. Transformants were selected on-
LB agar plates supplemented with 50 ,~Lg~ml ampicillin.
3 5 Transformants were analyzed for harboring the correct
recombinant plasmid using the alkaline lysis miniprep
t~ hn;qr1c. (Molecular Cloning) . Miniprep DNA was analyzed by
restriction enzyme mapping. 25 ml cultures of pRZ52 (CKB)
and pRZ53 (C~¢) were grown in LB with 5011g/ml ampicillin and
40 larger scale DNA preps were purified using QIAGEN plasmid
purification columns (QIAGEN Corp., Chatsworth, CA) .
Dideoxynucleoside chain-tF~rm;nAt;nn DNA secluencing reactions
WO 95112662 ~ PC~IUS94112624
2~7~a
were carried out according to the standard protocols
described in the Sequenase 2 O kit from USB tCleveland, OH).
[-35S]-dATP (~ew ~ngland Nuclear~ was used to radiolabel the
sequences for visualization on Kodak XAR film. Reactions
5 were separated through a 6% polyacrylamide gel, the gel
dried, and expbsed to ilm as described in Molecular
Cloning. The DNA sequence representing the coding regions
of CKs and CKM are in ~igure G1 and 2.
Once the CKM DNA sequence was ~lPtPrmin~9 to be
10 correct the a~npicillin resistance gene in pRZ53 was
exchanged for the kanamycin resistance gene from Tn903
~Nomura et al., ~ene, 3:39-51, 1987). The ampicillin gene
was cut out using the restriction enzymes Ssp~ and spml, and
the vector ends were blunt ended using T4 polymerase. The
15 kanamycin resistance gene had been cloned into the
polylinker in a pBR322 :vector. The gene was cut out of the
vector using BamH1 and was blunt ended with T4 polymerase.
The resulting C~M plasmid is referred to as pRZ69.
20 t`orc:truct; n~ the CRMl~ Co-P~ression Strain
pRZ52 and pRZ69 DNA was mixed together in a
concentration o~ approximately 50 llg/ml and 1 1ll of the
mixture electroporated into 20 1ll MClû61 cells. The cells
were plated onto 1~3 plates containing 50 llg/ml ampicillin
25 and 50 llg/ml kanamycin to select or cells that had been
co-tra~sformed with both plasmids. Co-transformants were
analyzed ~y restriction digestion of miniprep DNA (the
standard tP~ n; q~P is described in Molecular Cloning) .
WO 95112662 ~ 1 7 5 5 9 0 pC~ S94/12624
-- 7 --
p\nA 1 vr i n F~-~nressinn
Expression analysis of the co-transformants
was carried out as follows. Clones were grown overnight in
2 ml LB, 0.2~s glucose, 50 llg/ml kanamycin and 50 llg/ml
S i ~71l;n at 37 C shaking. In the morning 25 ml of LB with
50 ~Lg/ml kanamycin and 50 llg/ml ampicillin was inoculated
with 0 . 6 ml of the overnight culture and grown at 30 C
shaking. Cultures were grown to A600 about 0.4 then sampled
at one hour intervals for 4 hours. Cultures were left
10 growing overnight and an additional sample taken. Whole
cell samples were boiled in SDS-PAGE sample buffer and run
through 12% polyacrylamide gels to assess protein
production Gels were obtained f rom BioRad, and protocols
provided with gels were followed.
Several assays were used to ~F.t~rmin~ the level
and quality of the expressed r~ mhinAnt proteir,. Cultures
were grown as previously described. At A600 approximately
1. 0 or af ter overnight incubation cells were harvested and
resuspended in a lysis buffer (20 mM Bis-Tris pH 6.9, 0.259
20 Tween 20, 10 mM b ~ptoethanol, 10 mM EDTA, 10 mM EGTA,
lmM PNSF). The resuspended cells were lysed by sonication on
ice (or for larger scale analysis cells were lysed using the
Microfl~ ;7~r from Microfluidics Corp., Newton, MA ) and
the cell debris removed by centrifugation. Samples were
2 5 analyzed using the following assay systems after diluting
into lysis buffer. The Creatine Kinase Reagent (Sigma
47-UV) is a spectrophotometric assay for kinetic
determination of enzyme activity. The Creatine
Phosphokinase (CPK) Isoenzymes Kit (Sigma 715-EP) separates
3 0 the various isoforms (NM, MB, and BB) and stains for
activity. The CR-MB assay system for the Abbott ~c
analyzer uses a microparticle enzyme i oAqsay (MEIA) to
determine specific protein mass of CR-MB in a sample. (A11
protocols are provided with the assay kits.
:~uiv)- 1 ~nt ~
Those skilled in the art will recognize, or be
able to ascertain, using no more than routine
expGr;~ tAt;nn many equivalents to the specific embodiments
4 0 of the invention described herein. Such equivalents are
;nt.~n~ l to be ~- cced by the fallowing claims:
~O95/12662 j ' ~ PC~IUS94/12624 ~
2 ~ 8 -
SEQUENCE LISTING
( l ) GENE-RAL INFORMATION:
(i~ APPLICANT: Ziegler, Robin ~.
Lon~, Sue
( ii ) TITLE OF LNVI~ I'l'LUN: Method of Producing
Rl ' 'nAnt Dimeric Enzyme
l O
(iii) NU~3ER OF ~U N(.~ : 4
( iV) ~:U~ UNJ~;N~:~; ADDRESS:
(A~ ~nnR~. : Bill Gosz , Esq.,
1 5 Genzyme Corporation
(B) STREET: One Kendall Sc~uare
( C ~ CITY: Cambridge
( D ) STATE: MA
( E ) COUNTRY: U . S . A .
2 0 (F) ZIP: 02139
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CC~PUTER: 1:~ PC compatible
2 5 (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l . 0,
Version # l . 25
(vi) CURRENT APPLICATION DATA:
3 0 (A) APPLICATI~ NOMBER: US
( ~3 ) F ILING DATE:
( C ) CLAS S IF I CAT ION:
(viii) ATTORNEY/AGENT INFOR~TION:
3 5 (A) NAME: William G Go8z
(B) REGISTRATION NOMBER: 27,787
(C) ~ ~/DOCKET NOMBER: GEN3-l0.0
WO 95/126G2 217 ~ ~ ~ O PC~/IIS941~2624
_ 9 _
( ix) ~r~r ~ [ ~ ~ IN I ~ L l'ION INFORMATION
(A) ~rT T,T PT~nNF (617) 252--7583
(B) TELEFAX (617) 252-7600
( C ) TELEX 2 012 23 GENCA~B
( 2 ) INFORMATION FOR SEQ ID NO
(i) SEQ~ENCE CHaRACTERISTICS
(A) LENGTH 11~6 base pairs
(B) TYPE nucleic acid
(C) S'rR~NlJ~l~N~S`i single
(D) TOPOLOGY linear
( ii ) MnT F~'TTT~T. TYPE cDNA
(iii) ~YL~r~ L~lCAL NO
( iv) ANTI-SENSE YES
2 0 (v) FRAGMENT TYPE N-terminal
(vi~ ORIGINAL SOURCE
(A) OR~ANISM Homo sapiens
2 5 ( xi ) SEQUENOE L~ ClL~ l lON SEQ ID NO l
ATGCCCTTCT rr~ArAr.rrA CAACGCL~CTG AAr~rlrr~rr~rT L.~ W~ GGACGAGTTC 60
CCCGACCTGA r,rrrrrDrAA rAArrArATr~ GCCAAGGTGC ~rr.ArrrrrrA GCTGTACGCG 120
r-Arr-~Grr-rr~ rrAArAr~rAr r.rrrArrr~r TT,CACGCq'GG ACGACGTCAT rrAnArArrr 180
GTGGACAACC rr~r~rrArrr r.~rDrA~rA~r~ ACCGTGGGCT ~.1'1.1~:13 I~I.(j rr.Arr~Arr.Ar. 240
3 5 TCCTACGAAG TGTTCL~AGGA TCTCTTCGAC CCCATCATCG Ar~Arrrr.rA rrrr.rrir~rAr 300
- AAr.rrrArrn ATGACGACAA rArrrArrrr AArrrrr.ArA Arr~rrArr~ rrrrr.Arr.Ar 360
CTGGACCCC~ Ar~Arr~Tr~rT GAGcTcGr-GG GTGGCCACGG r~rrrrArrAT ~L~i~L . 4ao
i~.~LLC: rrr~r~r.rAr. rrrrrrr~An rr,rrr.AnrrA TCGAGAAGCT rrrrr~rrrAA 480
WO 95/12662 . ~ PC'FIUS9411262-1 ~
2 17 ~ 0
r~ rrrTrr-Drrr rr.ArrTrr.rr. GGCCGATACT ACGCGCTCAA GAGCATGACG ~40
~:Arrirrr.~r.r ArrDr~rDr~rT DTrr~ArnAr CACTTCCTCT Trr~DrAAr-rr ~ 600
5 ~l~ L~; rrTrr.r.f:rDT rrrrrrrr.Ar Tr~rrrr.Drr. rrr.rrrr.~AT rTrrrArDAT 660
r.ArDATAAr.A ~ l GTGGGTCAAC r.Arr.Arr.Arr ArrTrrr,r.~:T CATCTCCATG 720
r~r.AAr.rr~r7 rrAAr~Tr.AA GGAGGTGTTC ACCCGCTTCT r~rArrr~rrT CACCCAGATT 780
l O
GAAACTCTCT TrAAr.TrTAA GGACTATGAG TTCATGTGG,A ACCCTCDCCT GGGCTACATC 840
CTCACCTGCC CATCCAACCT rrr.rDrrr,r~ rTr,rrrr,r~r. GTGTCGATAT CAAGCTGCCC 900
AACCTGGGCA AGCATGAGAA GTTCTCGGAG GTGCTTAAGC GGCTGCGACT TCAGAAGCGA 960
nr~rDr~r~r~rG GTGTGGACAC ~l~ , TCGACGTCTC rD~rrr~TnAr 1020
~l~iW-r TCTCAGAGGT r~nAnrTr~r~Tr~ CAGATGGTGG 'rr~GACGGAGT rADrrTrrTr 1080
ATCGAGATGG AACAGCGGCT rr.Drr~rrr.r CAGGCCATCG ACGACCTCAT r~rrTr~rrrAr~ 1140
AAATGA 119.6
~IVO 95112662 2 1 7 5 ~ ~ ~ PC~IUS94l12624
1 ~ _
(2 ) INFORM~TION FOR SEQ ID NO: 2
( i ) SEQ~ENCE CH~RACTERISTICS:
~A) LENGT~: 1146 base ~airs
~B) TYPE: nucleic acid
~ C ) S'T'T~ A N I I ~ 'i 'i: s irlgle
~D) ~OPOLOGY: linear
~ ii) M~T,T~`TTTT~' TYPE: c~A
l O
(iii) ~Ul'~ll~AL: NO
(iv) P~NTI-SENSE: YES
(v) FRAG~ENT TYPE: N-ter~ninal
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
ATGCCATTCG r.~AArArrrA CAACAAGTTC AAGCTGAATT ArAAnrrTnA rr.Arr.Ar.~Ar 60
CCCGACCTCA rrAA~rA~AA CAACCACATG r~rrAAr~r TGACCCTTGA ACTCTACAAG 120
AAGCTGCGGG ArAArr.~r.A~ CCCPLTCTGGC TTCACTGTA~ ACGATGTCAT rrAr.ArAr.r.A 180
2 5 GTGGACAACC CAGGTCACCC CTTCATCATG ACCGTGGGCT ~L~L~ TGATGAGGAG 240
TCCTPLCGAAG TTTTCAAGGA ACTCTTTGAC CCCATCATCT CGGATCGCCA rrrr~rr~Ar 3 o O
AAArrrAr~r. ArAArrAr~ GPCTGACCTC AArrA'rGAAA ACCTCAAGGG TGGAGACGAC 360
CTGGACCCCA Ar~rArr.~rr~ rAr.rArrrrr, GTCCGCPLCTG rrrrrArrAq~ rAAr~rr~rAr 420
ACGTTGCCCC CACACTGCTC CCrTGGCGAG ~ TGGAGAAGCT rTr~r.~r~AA 480
3 5 r.r~r~rrAArA GCCTGACGGG CGAGTTCAAA rrr.AAr.~Ar~ ACCCTCTGAA r.ArrA~r.Arr. 5~0
rAr.AArr.Arr AGCAGCAGCT CATCGATGAC CACTTCCAGT TCGACA~GCC WL~ U~ 600
,~-~ CCTCAGGCAT rr,rrrrrrAr llr~r~rrrr~rr~ CCCCTGGCAT CTGGCACAAT 660
r.ArAAr~Ar.A ~7~lL~,l~il G~:GTGAAC GAGGAGGATC ACCTCCGGGT CATCTCCATG 720
W095112662 , PC~IUS9~112624
- 217~9a ~ 12-
r~r.~r~rr~ GCAACATGAA CCZ~_LLLL~ - ~.~ I 1.~ 1 I~ I GCGTAGGGCT GCAGAAGATT 780
GAG~AGATCT TTAAGAAAGC TrrrrD.rrrr TTCI!TGTGaA ArrDrrArr~r GGGCTACGTG 840
Sr~rsrr~rrr cATccAAr-cT r.rrr~r~r.rr. r~rrnTrr.~r. GCGTGCATGT GAAGCTGGCG 900
r~rrTr~r~r~ ~r~rrrr~ GTTCGAGGAG ATCCTCACCC 4~Ll~i~4L~ rr~r~r~r. 960
rrl~r~rr.~r. CGGTGGACAC AGCTGCCGTG rrr~rrl~r.~rAT TTGACGTGTC CAACGCTGAT 1~20
CGTCCGAAGT Ar.~r~rrl~r. CAGCTGGTGG TGaATGGTGT GAAGCTCATG 1080
GTGC~AA.;~TGG AGAAGAAGTT rr.~r~ rr~r CAGTCCATCG ~rr.P~r~r~ rrrrnrrr~r. , 140
I S AAGTAG 1146
WO 95/12662 2 t 7 5 5 ~ O PC~IUS9*12624
( 2 ) INFOKM~TION FOR SEQ ID NO: 3:
( i ) sEQr~ENcE rr~R ~
(A) LEWGTH: 381 amino acids
(B) TYPE: arnino acid
(C ) s~R~Nnr~r~NF ~.~: single
(D) TOPOLOGY: linear
(ii) Mr,r,r~(~rrr,r~ TYPE: protei~L
1 0
(v) FRAGMENT TYPE: N-terminal
(xi) sEslrJENcE L~ KLL~L~ L~I: SEQ ID NO:3:
1 5 Iet Pro Phe Ser ~sn Ser His Asn Ala Leu Lys Leu Arg Phe Pro Al~
5 10 15
Glu Asp Glu Phe Pro A~P Leu Ser Ala His Asn Asn His ~et Ala Lys
20 25 30
Val Leu Thr Pro Glu Leu Tyr Ala Asp Val Arg Ala Ly~ Ser Thr Pro
35 40 45
Ser Gly Phe Thr Leu Asp Asp Val Ile Gln Thr Gly Val Asp Asn Pro
2 5 so ss 60
Gly His Pro Tyr Ile Yo~ Thr V~l Gly Cys V.ll Ala Gly Asp Glu Glu
6s 70 75 80
3 0 Ser Tyr Glu V~l Phe Lys Asp Leu Phe Asp Pro Ile Ile Glu Asp Arg
85 90 95
His Arg Arg Tyr Lys Pro Ser AsP ASp Asp Lys Thr AsP Leu Asn Pro
100 105 110
Asp Asn Leu Gln Gly Gly A~p Asp . Leu Asp Pro Asn Tyr Val Leu Ser
115 120 125
4 0 Ser Arg Val Ala Thr Gly Arg Ser Ile Arg Gly Phe Cys Leu Pro Pro
130 135 140
WO 95/12C62 . ' ,` ' ` PCI!/US94112624~
- 21~9~ 14_
His Cys Ser Arg Gly Glu Arg Arq Ala Ile Glu Lys Leu Alo Val Glu
l~S lS0 lSS 160
Ala Leu Ser Ser Leu Asp Gly Asp Leu Ala Gly Arg Tyr Tyr Ala Leu
165 170 175
Lys Ser Met Thr Glu A1~ Glu Gln Gln Gln Leu Ile Asp Asp His Phe
180 185 190
0 Leu Phe Asp LYB Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala
l9S 200 205
Arg Asp Trp Pro Asp Al~ A121 Ars~ Ile Trp His Asn Asp Asn Lys Thr
210 215 220
Phe Leu Val Trp Val Asn Glu Glu Asp E~is Leu Arg Val Ile Ser Met
225 230 235 240
Gln Lys Gly Gly Asn Met Lys Glu Val Phe Thr Arg Phe Cys Thr Gly
2 0 245 250 255
Leu Thr Gln Ile Glu Thr Leu Phe Lys Ser Lys Asp Tyr Glu Phe Met
260 265 270
2 5 Trp Asn Pro His Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu Gly
275 280 285
Thr Gly Leu Arg Ala Gly Val Asp Ile Lys Leu l?ro Asn Leu Gly Lys
290 295 300
His Glu Lys Phe Ser Glu Val Leu Lys Arg Leu Arg heu Gln Lys Arg
305 310 315 320
Gly Thr Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe Asp Val
3 5 325 330 335
Ser Asn Ala Asp Arg Leu Gly Phe Ser Glu Val Glu Leu Val Gln Met
340 345 350
4 0 Val V~l Asp Gly Val Lys Leu Leu Ile Glu Met Glu Gln Arg Leu Glu
355 360 365
~ WO 95112662 ~17 ~ 5 9 0 rc~Nsg4/12624
~ 15 --
Gln Gly Gln Ala Ile Asp Asp ~eu D~et Pro Al~ Gln Lys
370 375 380
l O
1 5
~0
~Vo 95/12662 , ~ ' , PCFIUS94112624 ~
21~S5~ - t6-
( 2 ) rNFORMATION FOR SEQ m NO: 4:
( i ) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 381 a~ino acids /`
(B) TYPE: amino acid
(C) Slr1?1~N~ N~:~;';: single
(D) TOPOLO(~Y: linear
(ii) MOT T~'TIT,T. TYPE: protein
l O
(v) FR~T TYPE: N-terminal
(xi ) SEQUENCE L~ ~L~L~16~N: SEQ ID NO: 4:
Met Pro Phe Gly Asn Thr His Asn Lys Phe Lys Leu Asn Tyr Lys Pro
5 10 15
Glu Glu Glu Tyr Pro Asp Leu Ser Lys His Asn Asn His Met Al~ Lys
20 25 30
Vel Leu Thr Leu Glu Leu Tyr Lys Lys Leu Arg Asp Lys Glu Ile Pro
35 40 45
Ser Gly Phe Thr Vzll Asp Asp VP1 Ile Gln Thr Gly V~l Asp Asn Pro
2 5 so s5 60
Gly His Pro Phe Ile Met Thr Val Gly Cys Val Al~ Gly Asp Glu Glu
6s 70 75 80
3 0 ger Tyr Glu VP1 Phe Lys Glu Leu Phe Asp Pro Il~ l:le Ser Asp Arg
85 90 95
His Gly Gly Tyr Lys Pro Thr Asp Lys His Lys Thr Asp Leu Asn His
100 105 ~ 110
Glu Asn Leu Lys Gly Gly Asp Asp Leu Asp Pro Asn Tyr V~l Leu Ser
115 120 125
Ser Pro V- 1 Ar~ Thr Gly Arg Ser Ile ~ys Gly Tyr Thr Leu Pro Pro
4 0 130 135 140
WO95112662 2~7~59D PC~IUS94112624
His Cys Ser Arg Gly Glu Arg Arg Ala Val Glu Lys Leu Ser Val Glu
145 150 155 160
Ala Leu Asn Ser ~eu Thr Gly Glu Phe Lys Gly Lys Tyr Tyr Pro Leu
165 170 175
Lys Ser Met Thr Glu Lys Glu Gln Gln Gln Leu Ile Asp Asp His Phe
180 185 190
0 Gln Phe Asp Lys Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala
195 200 205
Arg His Trp Pro Asp Al~ Pro Gly Ile Trp His Asn Asp Asn Lys Ser
210 215 220
l S
Phe Leu Val Trp Val Asn Glu Glu Asp His Leu Arg Val Ile Ser Met
225 230 235 240
Glu Lys Gly Gly Asn Net Lys Glu Val Phe Arg Arg Phe Cys Val Gly
2 0 2~5 250 255
Leu Gln Lys Ile Glu Glu Ile Ph~ Lys Lys Ala Gly Hia Pro Phe Met
260 265 270 ~~
2 5 Trp Asn Gln His Leu Gly Tyr Val Leu Thr Cys Pro Ser Asn Leu Gly
275 280 285
Thr Gly Leu Arg Gly Gly Val His Val Lys Leu Ala His Leu Ser Lys
290 295 300
His Pro Ly~i Phe Glu Glu Ile Leu Thr Arg Leu Arçr Leu Gln Lys Arg
305 310 315 320
Gly Thr Gly Ala Val Asp Thr Ala Ala Val Gly Ser Val Phe Asp Val
3 5 325 330 335
er ~sn Ala Asp Arg Leu Gly Ser Ser Glu Val Glu Gln Val Gln Leu
340 345 350
4 0 Val Val Asp Gly Val Lys Leu Met Val Glu Met Glu Lys LYs Leu Glu
355 360 365
WO 95/12662 , ~ PC~IUS94/;2624
21~5~a - 18 -
Lys Gly Gln Ser Ile Asp Asp Met 'fle Pro ~1~ Gln Ly~
370 375 380
