Note: Descriptions are shown in the official language in which they were submitted.
1341633
TITLE OF THE INVENTION
INTERLEUKIN-2 POLYPEPTIDES
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to interleukin-2
polypeptides.
Brief Description of the Prior Art
Interleukin-2 (hereinafter referred to as "IL-2"),
formerly referred to as T cell growth factor, is a soluble
protein, and is produced from T cells activated with a
lectin or an antigen (Morgan, D. A. et al. Science, 193,
1007-1008 (1976), Gillis, S. et al., J. Immunol., 120,
2027-2033 (1978). Interleukin-2 (IL-2) is capable of
modulating lymphocyte reactivity and promoting the in vitro
long-term culture of antigen specific effector T-lymphocytes
(Gillis, S. et al., Nature 268, 154-156 (1977)). IL-2 is
also known to manifest other relevant biological activities
such as enhancement of thymocyte mitogenesis (Chen, B. M.
et al., Cell. Immunol., 22, 211-224, (1977), Shaw, J. et al.,
J. Immunol. 120, 1967-1973, (1978)), induction of cytotoxic
T cell reactivity (Wagner, H. et al., Nature, 284, 278-280,
(1980)) and anti-SRBC plaque forming cell responses (Gillis,
S. et al., J. Exp. Med., 149, 1960-1968, (1979)) in cultures
of nude mouse spleen cells. Accordingly, this lymphocyte
regulatory substance is useful in potentiating humoral and
cellular immune responses and in restoring immune deficient
state to a normal humoral and cellular immune state. These
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1341633
identified immunological activities of IL-2 indicate that
IL-2 is useful for medical immunotherapy against immuno-
logical disorders including neoplastic diseases, bacterial-
or viral infections, immune deficient diseases, autoimmune
diseases etc. (Papermaster, B. et al., Adv. Immunopharm.,
507, (1980)).
SUMMARY OF THE INVENTION
Now, new IL-2 polypeptides, which bear threonine
as the C-terminal amino acid and no sugar moiety, have been
found in the cells of Escherichia coli, which has been
constructed by incorporation into a host of Escherichia coli
of a recombinant DNA possessing a DNA fragment coding for
IL-2 polypeptide.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing no sugar moiety comprising -
culturing, in a culture medium, prokaryotic cells
transformed with a DNA sequence encoding interleukin-2-
polypeptide,
recovering interleukin-2-polypeptide, and
purifying the recovered interleukin-2-polypeptide,
said DNA sequence comprising a gene encoding a
polypeptide which possesses a biological activity of human
interleukin-2, wherein the DNA sequence being present on a
plasmid is capable of replicating in said cell, said gene
having a coding sequence located at a position downstream of
a promoter sequence.
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In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing threonine as the C-terminal amino acid and no sugar
moiety comprising culturing cells of Escherichia coli
constructed by gene-recombination in a nutrient medium, said
cells being capable of producing said interleukin-2-
polypeptide, and recovering said polypeptide from said
medium, said cells being transformed with a DNA sequence
comprising a gene encoding a polypeptide possessing
biological activity of human interleukin-2 and being present
on plasmid pTIL-2-21 or plasmid pTIL-2-22, wherein said
plasmid is capable of replicating in said cell, and said
gene has a coding sequence located at a position downstream
of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing no sugar moiety comprising (i) culturing, in a
culture medium, prokaryotic cells transformed with a DNA
sequence encoding 132 consecutive amino acids of the
interleukin-2-polypeptide as set forth in Figure 2, (ii)
recovering interleukin-2-polypeptide, and (iii) purifying
the recovered interleukin-2-polypeptide, said DNA sequence
comprising a gene encoding a polypeptide which possesses a
biological activity of human interleukin-2, wherein the DNA
sequence being present on a plasmid is capable of
replicating in said cell, said gene having a coding sequence
located at a position downstream of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing no sugar moiety comprising (i) culturing, in a
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1 0 3 4 1 633
culture medium, prokaryotic cells transformed with a DNA
sequence encoding 133 consecutive amino acids of the
interleukin-2-polypeptide as set forth in Figure 2, (ii)
recovering interleukin-2-polypeptide, and (iii) purifying
the recovered interleukin-2-polypeptide, said DNA sequence
comprising a gene encoding a polypeptide which possesses a
biological activity of human interleukin-2, wherein the DNA
sequence being present on a plasmid is capable of
replicating in said cell, said gene having a coding sequence
located at a position downstream of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing no sugar moiety comprising (i) culturing, in a
culture medium, prokaryotic cells transformed with a DNA
sequence encoding interleukin-2-polypeptide as set forth in
Figure 2, said interleukin-2 polypeptide bearing alanine,
proline or methionine as the N-terminal amino acid, (ii)
recovering interleukin-2-polypeptide, and (iii) purifying
the recovered interleukin-2-polypeptide, (iv) said DNA
sequence comprising a gene encoding a polypeptide which
possesses a biological activity of human interleukin-2,
wherein the DNA sequence being present on a plasmid is
capable of replicating in said cell, said gene having a
coding sequence located at a position downstream of a
promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
bearing no sugar moiety comprising (i) culturing, in a
culture medium, prokaryotic cells transformed with a DNA
sequence encoding interleukin-2-polypeptide, (ii) recovering
interleukin-2-polypeptide, and (iii) purifying the recovered
interleukin-2-polypeptide, said DNA sequence comprising a
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13 41 633
gene encoding a polypeptide which possesses a biological
activity of human interleukin-2, wherein the DNA sequence
being present on a plasmid is capable of replicating in said
cell, said gene having a coding sequence located at a
position downstream of a promoter sequence and wherein said
interleukin-2-polypeptide has the following amino acid
sequence -
X-Tlir See See ;e1 The Lv ; lye r GIN Lou 031N X'oU
Gin Hie Len Lea Lou Asp Lou CAN Met:. ' 1 ? e . Leta. A e W .I1' 1 e
Ac;N Ass 'Tyr L,y f#sNN) A.rg Mat T,ou The Ptrry
Lys Plie '1'e lfet-. ee Lys Lys Ala The C1.u Lou Lys ..ji ZL(J_
91N (.'Ys -Lou Gin G.lt.z' G7.t~ 11e JL ,. r-c Let tt w7.-x'3. T. u
eN r t ii raLle CIZ Be). Lys As,P f'lat` Pie ou Arcj Pro A:rrq \.s t}
Lou :1.J_e See A tN I n 1rat<s sl 1:i ' Val Lou G1tt :.en L e i_i y
Se till! "1:'112 ie Phe M L' Cy; (;3,u1 '11y1r' i- 1a 1 sp L1.{_). '1`37y Ais
1'l r. I: 1.e Val G]lr I,~__r; ! eN Arc T' .r ~ l lc "t.'fl.r.' 1?3le Cyr
t"r:lt
See lie I.1~- Ser `.~L12e Len Tfz.i
t,rli reiy rf preset~tt;, Pro, Ata-Pro, MOt. P o or M1et- A1.s
l~xo.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
as set forth in Figure 2 bearing threonine as the C-terminal
amino acid and no sugar moiety, comprising culturing cells
of Escherichia coli constructed by gene-recombination in a
nutrient medium, said cells being capable of producing said
interleukin-2-polypeptide, and recovering said polypeptide
from said medium, said cells being transformed with a DNA
sequence comprising a gene encoding a polypeptide possessing
biological activity of human interleukin-2 and being present
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13 41 633
on plasmid pTIL-2-21 or plasmid pTIL-2-22, wherein said
plasmid is capable of replicating in said cell, and said
gene has a coding sequence located at a position downstream
of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
having 132 consecutive amino acids of the polypeptide as set
forth in Figure 2 bearing threonine as the C-terminal amino
acid and no sugar moiety, comprising culturing cells of
Escherichia coli constructed by gene-recombination in a
nutrient medium, said cells being capable of producing said
interleukin-2-polypeptide, and recovering said polypeptide
from said medium, said cells being transformed with a DNA
sequence comprising a gene encoding a polypeptide possessing
biological activity of human interleukin-2 and being present
on plasmid pTIL-2-21 or plasmid pTIL-2-22, wherein said
plasmid is capable of replicating in said cell, and said
gene has a coding sequence located at a position downstream
of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
having 133 consecutive amino acids of the polypeptide as set
forth in Figure 2 bearing threonine as the C-terminal amino
acid and no sugar moiety, comprising culturing cells of
Escherichia coli constructed by gene-recombination in a
nutrient medium, said cells being capable of producing said
interleukin-2-polypeptide, and recovering said polypeptide
from said medium, said cells being transformed with a DNA
sequence comprising a gene encoding a polypeptide possessing
biological activity of human interleukin-2 and being present
on plasmid pTIL-2-21 or plasmid pTIL-2-22, wherein said
plasmid-is capable of replicating in said cell, and said
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13416;3
gene has a coding sequence located at a position downstream
of a promoter sequence.
In accordance with the invention, there is
provided a method of producing an interleukin-2-polypeptide
as set forth in Figure 2 bearing threonine as the C-terminal
amino acid, bearing alanine, proline or methionine as the N-
terminal amino acid and no sugar moiety, comprising
culturing cells of Escherichia coli constructed by gene-
recombination in a nutrient medium, said cells being capable
of producing said interleukin-2-polypeptide, and recovering
said polypeptide from said medium, said cells being
transformed with a DNA sequence comprising a gene encoding a
polypeptide possessing biological activity of human
interleukin-2 and being present on plasmid pTIL-2-21 or
plasmid pTIL-2-22, wherein said plasmid is capable of
replicating in said cell, and said gene has a coding
sequence located at a position downstream of a promoter
sequence.
In accordance with the invention, there is
provided an interleukin-2-polypeptide bearing no sugar
moiety, which is produced by culturing prokaryotic cells in
a culture medium, transformed with a DNA sequence encoding
interleukin-2, recovering the produced interleukin-2,
purifying the recovered interleukin-2; said DNA sequence
comprising a gene encoding a polypeptide which possesses a
biological activity of human interleukin-2 and being present
on plasmid pTIL-2-21 or plasmid pTIL-2-2, wherein said
plasmid is capable of replicating in said cell, and the
coding sequence of said gene is located at a position
downstream of a promoter sequence.
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13 4 1 633
In accordance with the invention, there is
provided an interleukin-2-polypeptide bearing no sugar
moiety and having 132 consecutive amino acids of the
polypeptide set forth in Figure 2, which is produced by
culturing prokaryotic cells in a culture medium, transformed
with a DNA sequence encoding interleukin-2, recovering the
produced interleukin-2, purifying the recovered interleukin-
2; said DNA sequence comprising a gene encoding a
polypeptide which possesses a biological activity of human
interleukin-2 and being present on plasmid pTIL-2-21 or
plasmid pTIL-2-22, wherein said plasmid is capable of
replicating in said cell, and the coding sequence of said
gene is located at a position downstream of a promoter
sequence.
In accordance with the invention, there is
provided with an interleukin-2-polypeptide bearing no sugar
moiety and having 133 consecutive amino acids of the
polypeptide set forth in Figure 2, which is produced by
culturing prokaryotic cells in a culture medium, transformed
with a DNA sequence encoding interleukin-2, recovering the
produced interleukin-2, purifying the recovered interleukin-
2; said DNA sequence comprising a gene encoding a
polypeptide which possesses a biological activity of human
interleukin-2 and being present on plasmid pTIL-2-21 or
plasmid pTIL-2-22, wherein said plasmid is capable of
replicating in said cell, and the coding sequence of said
gene is located at a position downstream of a promoter
sequence.
In accordance with the invention, there is
provided an interleukin-2-polypeptide bearing no sugar
moiety as set forth in Figure 2 and bearing alanine, proline
or methionine as the N-terminal amino acid, which is
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X 3 41 633
produced by culturing prokaryotic cells in a culture medium,
transformed with a DNA sequence encoding interleukin-2,
recovering the produced interleukin-2, purifying the
recovered interleukin-2; said DNA sequence comprising a gene
encoding a polypeptide which possesses a biological activity
of human interleukin-2 and being present on plasmid pTIL-2-
21 or plasmid pTIL-2-22, wherein said-plasmid is capable of
replicating in said cell, and the coding sequence of said
gene is located at a position downstream of a promoter
sequence.
In accordance with the invention, there is
provided an interleukin-2-polypeptide bearing no sugar
moiety, which is produced by culturing prokaryotic cells in
a culture medium, transformed with a DNA sequence encoding
interleukin-2, recovering the produced interleukin-2,
purifying the recovered interleukin-2; said DNA sequence
comprising a gene encoding a polypeptide which possesses a
biological activity of human interleukin-2 and being present
on plasmid pTIL-2-21 or plasmid pTIL-2-22, wherein said
plasmid is capable of replicating in said cell, and the
coding sequence of said gene is located at a position
downstream of a promoter sequence and wherein said
interleukin-2-polypeptide has the following amino acid
sequence -
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13 41 633
X-`I'ihr. Ser Sea: Cer Thr. I,W; Lys . 't'h,r GIN Lou C t4 Lou
GJ.u His Lou Lou Lou Asp Lou GIN Met '1]e Let). AsN Ã.. l,t! .F, 1-o
A :N AsN Tyr T y rtsN 1'r.o ,v Lee Thr Aru Met Lee Phr
1'..,y4 Phe Trey MeL Pro Lys Lys Ala rfhr GIii.. Lett Lys His Let`
G1.N CCs Leu Givi Glt.z Glu bet, : y Prr-n Lou Glrl C..1.u Fa}. eu
tsN 1,c t:u Al e CGli Sex- Lys ~tsP i1Jie W-8 Lou Arg Pro Arcj ,1, s3-,
T,ee Ile scar Asir Ile Ash Vel le Val Leon J z'
7F-:r u r 1111':r' Pht;. Met C1. to Tyr `l1 a. Asp `(,`hr h-1 a
"1.'1 7:` .l I e Val (1 ti )?I-)e 1,ee faeN Arc ".rtp :Cie. '.k' he Phe Cy-3
("l (q
Se l ie t 1 bier 1`Ji~.` Lee Thr
wherein 7t repzeset t5 Pro, is-PPro, Mot--pro or Met--Ma-
In accordance with the invention, there is
provided a human interleukin-2-polypeptide preparation
having a specific activity of about 5 x 10' units/mg,
wherein the polypeptide bears threonine as the C-terminal
amino acid and no sugar moiety.
In accordance with the invention, there is
provided a human interleukin-2-polypeptide preparation
having a specific activity of about 5 X 107 units/mg,
wherein the polypeptide as set forth in Figure 2 bears
alanine as the N-terminal amino acid and no sugar moiety.
In accordance with the invention, there is
provided a human interleukin-2-polypeptide preparation
having a specific activity of about 5 X 10' units/mg,
wherein the polypeptide as set forth in Figure 2 bears
proline as the N-terminal amino acid and no sugar moiety.
2h -
134163,
In accordance with the invention, there is
provided a human interleukin-2-polypeptide preparation
having a specific activity of about 5 X 10' units/mg,
wherein the polypeptide has no sugar moiety and the
following amino acid sequence I:
ciro AC .;d Sequence i
Ala I'i, ) Thr Ser St Ser Tlar Lyr Lys Thr GZNN Leo GIN LLeo
G.lu Ftls L u Leu Leo Asia Leu GIN M t Iie Leo AsN Gly I.1e
Asti AsN Tyr Lys As1.4 Pro Lys Leo T~I-ir Arg Met Leo fir rlye
Lys Phe Tv x, Iliet Pica Lys Lys Ala Tiir Glu Lau Ly'3 1iio Lcu
GIN Cys Lets Glu Glu C,lu fõeu t,yo Pro Leu G1u C1u Val Leu
AePN Leo Ala GIN Ser Lye AsNN P tie His Leu Avg Pro Arg Aso
t..eu ! l e Ser Ask l i p Asti Vol 1,10 Vol Leo Glu L eta t. yu y
Star Glu fir thy Pile lie t; Lys Glta Tyit Alga Aeel> 01u I., r Alaa
~l~r Ile Vol Cl.u Phe Leu AsN Artj Trp 11.e Thrr PhL- Cye GIN'
Stec TIn lIe Ser r Leu Tli
In accordance with the invention, there is
provided a human interleukin-2-polypeptide preparation
having a specific activity of about 5 X 107 units/mg,
wherein the poly-peptide has no sugar moiety and the
following amino acid sequence II:
A,aancr Acid Sequence 11
pro Tllr 5ar Sar~ Ser Thy Lys Lys Thr GIN Leo GiN Lieu Cl.u
}4I~t tear i eu L u Aep Leu GIN M t ble Leta AsH Gly lie A:~H
AIN Tyr Lys AsW pro Lye L.t.us, tl.r Ar9 diet; Leo Thr P1le Lyrr
Ph,e. Tyt NeL l'1'~, Lys ye+ Ala ihr G1u Lau Lys H s l:eo G.1N
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13 41 633
Cy Y [ e.~ Clu ts1.F C 1u LLuu [=Y: u [sir 11u C;1~, Val 1,t h !
r ,~ 1 r C: ,l-r r I L) A., N Plat, if .i.-; i=. a u A r rj P Tr d Arc, Asp
1. u
IJ 5cr , st lie As 1131 11D V I Le u tlIw U w Lys Cly
G.lu Thr Thr Phe i4et Gys Glu Tyr Ala Asp C!.u Thr Al, , Th:
IJ.ta V11 G ;a the L J A!U Al'c Yr,p lltt Thr P'he Cy`> CI*l Set`
1 AC I1r, $r,,: Thr i, e:ii =i`ihr
In accordance with the invention, there is
provided an interleukin-2-polypeptide preparation bearing
threonine as the C-terminal amino acid and no sugar moiety,
having biological activity of human interleukin-2 and having
the amino acid sequence:
X-- Thr- Sr.>.r. Ser 1 cr: Phhi L r, Lys `.It'hr (1N Leu 0111 Leu
C3l11 His Le .u Lou beet Asp Leo GIN Met lie Lou sN Gly lie
AsN Asi.4 Tyt:' Lys Asi"E Pro Lys Leu Tar Arch Met: lee Phi: 1?he
Lys Phe Tyr Met Pro Lyre Lys TUe '&'I'tr Gb~t Lett Lys His I,cu
G .N Cys Leta CCl.u CY u Glu Lou Lys Pro Lcyta Glu GIc Vai Lou
Asti lieu Ala GIN er Lys Asti Pl-ho R is Leo Are Pro Are Asj
.,cyan T1t er_ AsN I:1is As C! \ai lte Val Let. Gl i Lett t.yF; 1y
seat G1.L1 T'hr Tat- 1-'lto Met Cys Glu Tyr Ale hasp Glu "i."lki Ata
Thr Ito Val C lA Plhe Leu AsW Arq T : p 'Cie. `;k"hr Plac7~ Cys (31 N
Ser V.":= lie firer; `I'hr Lou Thr
wherr_:s x X. r:ep)a:esexats Pr:o, A.ie' Prrc>, ?,Jet-pro or Met,-Al.a--
Pr o.
In accordance with the invention, there is provided a
pharmaceutical composition, comprising the interleukine-2-
2j -
X341633
polypeptide preparation as described herein, in association
with a pharmaceutically acceptable carrier.
In accordance with the invention, there is
provided a plasmid comprising a DNA sequence coding for a
human interleukin-2-polypeptide obtainable from a
transformant selected from FERM BP-225, FERM BP-226, FERM
BP-248, FERM BP-249 or FERM BP-245.
In accordance with the invention, there is
provided a an interleukin-2-polypeptide as described herein
for use in medical immunotherapy against immunological
disorders.
In accordance with the invention, there is
provided an interleukin-2 polypeptide preparation as
described herein for use in medical immunotherapy against
immunological disorders.
In accordance with the invention, there is provided a
pharmaceutical composition as described herein for use in
medical immunotherapy against immunological disorders.
In accordance with the invention, there is provided an
interleukin-2-polypeptide as described herein for use in
treatment of neoplastic diseases, bacterial or viral
infections, immune deficient diseases and autoimmune
diseases.
In accordance with the invention, there is provided an
interleukin-2 polypeptide preparation as described herein
for use in treatment of neoplastic diseases, bacterial or
viral infections, immune deficient diseases and autoimmune
diseases.
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In accordance with the invention, there is
provided a pharmaceutical composition as described herein
use in treatment of neoplastic diseases, bacterial or viral
infections, immune deficient diseases and autoimmune
diseases.
In accordance with the invention, there is
provided a n unglycosylated polypeptide preparation
possessing human interleuking-2 activity and having a
specific activity of not less than 104 U/mg, whre said
polypeptide is characterized by the amino acid sequence:
X1a,Ea-Pea-Tt~r-Sir,~Ã:e=~er-TH~rmLys_E_~~..ft '-~Hr~ Let~=-GHrmL~c~-t~lu-k#Hs-
Lr~u~~.eu=L~u~t~s~..
La~f GIn tvlet=-Hle-Le f Asn-GJyf~ilc~-A ?~~ Asn - "ye Lys4A n==Pro 1.yfs4 eu
~'hr-Ar' Met-l_FS ~,
Thr-H he-Lys--Pha-Tyr- at-Pro-Lys-E y,s-AEa- hrGHu-Lou-LYs FHis-Lou cin i ys
Leu 1
bf== fu-Lou, Lys-Wo_j-.,etf_ lu-Glu-Vfal-Leu-=.As -,Lau,AIa-G1r~ . ee~! )'s .
s i tah ~# Hs L ;
Ary-P~~~a-,dry-.'-tsp~Leu-EHe-der-Asr~ll4-Asn=L~aH-I(r.-1~~~l-Lc=r~,s ~lu-
Le~fnLy~-Gly-,~ee=i;lu-
t._Ãir F tr~#~t~ ~f ~t ys C~Hu 1 yxr Ai~a ks ~ t:3lu ihr ~'y14 Thr EJe- Va!
Ofu PHf ..~_e~a % r~ , r~..
t"+p~fHr f hrl'h~ c ys t~I~~~r lie Hle~e~ ~I"hr Leu .I-.ttr -
and X. .i. ; t'S e f.-. or
H .
In accordance with the invention, there is
provided a pharmaceutical composition comprising (a) a
pharmaceutically acceptable carrier, vehicle or diluent and
(b) an unglycosylated polypeptide preparation possessing
human interleukin-2 activity of not less than 104 U/mg,
where said polypeptide is characterized by the amino acid
sequence:
X /~Ha_Pea-=I'hr-Sei ~ er==~~r~Thr~Lys-Lys=-Ttll`-G In = Le~~=-Gln--Lou-Glu-
t{Hs~1.eu~L4u-L~u~~,s~-
Loti-GIn-Met., HIe-Leu-Ast1-Gly-tlc-f,sn-Asn-'t yr-=Lys-A:sn-Pro-Lys-Leu-Thr-
Argp iet-Le(I-
Thr-Plie-Lys--F he-Tyr-fviet-Prey-Lys-E-ys-Ala-"Fhr-Clu-L.eu-l..ys.-H-Iis-Lou-
=Gin-Cys-Leu-Blu-
CH~f=-f3Hu-LEU-=L}+s-F'ro-Leu-Glu -GHu-Vt~l==Leu-~t~ri==I_eu-~f~==Glrr-er'~Lys-
/~sr~A#'ho~~it's-Leu-
Arrg-Pro-Arg-Asp-Le=u-ile-Ser.-Asn-lie-Asti-Vai-II -1Eal4L u-t_ I~f-Leu L_y5-
Gly-Ser GIu
t hr Thr~Ph -( t Cy~s-CHu Tyr A3 Asti Glu 1`I~r 'til TH r EIe~V I f Htj-Pile-
Leu-Asti-Arl--
Tt}~-lia--i'lfr-F'l?~-C:yrs-Gtr nor-Hle-tle-.~e~--1'l7r Lefa-,-,hr
and X i.; Mot . or H.
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1341633
In accordance with the invention, there is
provided a method for preparing a pharmaceutical composition
comprising (a) a pharmaceutically acceptable carrier,
vehicle or diluent and (b) an unglycosylated polypeptide
preparation possessing human interleukin-2 activity of not
less than 104 U/mg, where said polypeptide is characterized
by the amino acid sequence:
X<=Ala-Pra)-Thf-Ser-Stir-;ter-'t hj'-Lys- LysJhr-GIn-Leto-GIn-Leu-Glu-!={i&Leu-
Leta-LOW-, s -
twatr-Gln-;lvlt #llc-Letr-Asn-Gly-llt~-A:;n-Asr~-- ~ yr=LyS~Ast~-Prr~-Lyps-
L.eu~ThrArt~w~jet==I -txtÃ..
Thr-Ptte,I~ys-PfieJyr-Met-Pto-Lys-I_y-5-AIa-Thr-G1u-Leu-Ly -Hfs-LeU-GIn-
Cys,L:etj-(3 It,-
1Ir1==C l' U-1- BU-Lys- I o-Lo i- GJU-Glu-V Ãl-Lcu- `tsn-Lear-Na==GIn-Ser-I-
ys=-Asn-Phe- H s-Leu-
ArO-Prt -Ary=-Asp-l_t u-Ile-Ser-Asn-11Ie-Asn-Val-Ile-Val- Leu- lwLeu-Lys-Gty-
Saj%GJu
iF"art'-~"krr~f~he~J~fief-r ~y5-GIu-Tyr==fiJa-Asia-Glu-l-I~r' ,r~l~~-"I'hr-
Ili..l(~I-C~[tr-I'I~r;-l..era-~~sn~,~rc~_.
TrP lle-Thr t l-c Cys-Gin- t r= lit-Ile St r= I'I-r Leta-Thr,
arid X. i ' ,;Fiat or H .
comprising admixing said polypeptide preparation with said
carrier, vehicle or diluent.
In accordance with the invention, there is
provided a pharmaceutical composition comprising (a) a
pharmaceutically acceptable carrier, vehicle or diluent and
(b) an unglycosylated polypeptide preparation possessing
human interleukin-2 activity of not less than 104 U/mg,
wherein said polypeptide is characterized by the amino acid
sequence:
X~ltfa-fi~ro--CI~r-Ser-S~;r-==~~r-`fhr-l.ys-t,ys=--~"f"7r-Gtn
I..etx==Gtrt,lneu Glu-#"{ts-l.eu-0L.r;u,t~eu~As~
Lau-GJn-Met-11o-Leu-Asn-Gly-lle-Asr I-As r7-'Tyr,.L.yfs-Asn-P(0-LyFs-Leu-Thr-
Arg-Met-Lett-.
Thr-Phe-Lys--Phe-Tyf-Met-No-Lys-Lys -Ala 'l #tr-C,lu Lcart l.:ys k~rs eu Gin-t
yes-Leu- ir,r-
Tlr-=t lu-l eu Lys-l rc -L.etrNGJu-Gfu-Val-Leu-,Asn-,L e u-Ala-GinMSe r'-L.ys=-
Asn-Phe-H s-Leu-
Ar-Pry-Ark-Asp~Le=u-ilenSer-Asr~==JJe-,gsn~Val-Ile-VII-Ler.r-C~lu~L+~r~l_ys-
GJy-Ser=
Thr'-Thr-Prie-Met- `ys-Gtu-Tyr-Ala-Asp- Flu-Thr'Ain-Thr-I1 ~V 1 C Iu~F't~n
I..era~A;n Ark--
TrP lle-Thr-- hc-Cys Gbi. er ile-Ile Ser-Ti=rr Leta=_i hr,
and Y. i ; ri t. or H.
for the treatment or prevention of neoplastic disease.
2m -
13 41 633
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising a sequence
Met-TyrwArg-Met-G1n-teu--Leu-Si r-Cys-I1e-A1a-Leu.-Sex-
Leu-A1a-Leu-Va l-Thr-Asn-Ser--Ala-Pro-`I'hr-Ser-Ser-Ser-
Thr-Lys-Lys--filar-Gl.n-Leu-Gl n-Leu-Glu-His-Leu-Leu--Leu-
Asp-Leu-G1n-Met--lie-Leu-Asr1-Gly-Ile-A, n--Asn-Tyr-Lys-
Asn-Pr:o-Lys -Leu--Thr-Arg -Met-Leu-Thr-Phe-Lys--.Pete--Tyr-
Met-Pro-Lys-Lys-.Aia-Thr-G1u- Ler -Lys-His--Leu-Gln-Gys--
Leu-Glu-Giu-Glu-Leu-Lys-Pro-Leer--G1u-Gl.u-Va1-Leu-Asz -
Leu-Al.a-Gln-Ser-Lys-Asn-Phe-His-Leu-Arg-Pro-Arg--Asp-
Leu-Ile-Ser -Asn-Ile-Asn-Va.l.-ll.e--Val-Leu-Giu--Leu-Lys-
Gl.y-Ser-Glu--Thr-Thr-Phe-Met-Cys--Giu-Tyr-Aia-Asp-Gl.u-
TI}r-Ala-Thr-Iie-Vai-G.lu-Phe-Leu-wAsn-Arg-Trp-Ile-Thr--
Phe-Cys4Gin-Ser-lie-Tie-Ser--Thr:-Leu-Thr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising a sequence
X-Thr-Ser-Ser-SereNThr--Lys-[ yS-Thr-G1n--Leu-Gln-Leu-Giu-
His-Leu-Leu -Leu-Asp-Leu- G1n-MetM-1Ie-Leu-Asn-Gly-Ile-
Asn_A,sn-Tyr-Lys_ASn-Pro-Lys -Leu-Thr-Arg,7Met~Leu- `hr-
Phe-Lys--Phe-Tyr--Met-Pro-Lys-Lys-Ala--Thr-G1u-Leu-Lys
His-Leu-Gln-Cys -Leu-Giu-Glu-Giu-GeuY-Lys-Pro-Leu.-=Glu-
Giu-Val-Leu-Asn-Leu-Al.a-Gln-Ser -Lys=-Asn-Phe-His-Leu-
Arg-ProArg-Asp-Leu-11e-Ser--Asf-Iie-ASn-V'ai-lle-Val-
Leu-Glu-Lau-Lys-Giy-Sex--Gl.u- hr--Tlir--Phe-Met-CCs-Gi.u-
'Syr-Al.a-Asp -Gl.u-Thr-Al.a-Thr-Il.e-Val -Giu-Phe-Leu..-Asn-
Argw-Trp-Il.e-Thr--Phe-Cys-Gln-Ser-Il.r --lle--Ser-Thr-Leu-,
Thr
2n -
13 41 633
wherein X represents Ala-Pro, Pro, Met-Ala-Pro or Met-Pro.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising a sequence
X Ala-lira-Thr-Ser-Ser-Set-Thr-Lys-Lys-Thr GIn-Leu-Gin-Leu-Glu-His-Leu-Leu-Leu-
Asp-
Leu-Gln-Met-lle Leu-Asn-Gly-lle-Asn-Asn-T"yr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-
Leu-
Thr-Phe-Lys-Phe-Tyr-Me#-Pro-Lys-Lys-Ala-Thr-Glu-Leu-Lys-Hls-Leu-c ln-Cys-Leu-
Glu-
Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-l ou-Asn-Leu-Ala-Gln-oOer-Lys-Asn-Phe-His-
Leu-
Arg-Pro-Arg-Asp-Leu-Ile-Ser-Asn-iie-Asn-Val-ile-Val-Leu-Clu-Leu-Lys-G ly-Ser-
Giu-
Thr-Thr-Phe-Met-Cys-Glu-Tyr-Ala-Asp-Glu-Thr-Ale-Thr-lla-Val=Glu-Phe-Leu-Asn-
Arg-
Trp-lie-Thr-Phe Cys-Gin-Ser-ilo-lie-Ser-Thr-Loci-Thr,
wherein X is Met or H.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising the sequence shown in
Figure 2.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising the following
sequence
-
2o
1341633
Ai it Acid Sequence l
Ala Pro Thr Ser Sex Se: Th-r Lys' Lys Thr GIN Lcu GIN- Lt,.,j
Glu Ki3 Leu Leu Lcu Asp Lew GIN Met Iie t.eu A 5N Gly Ile
ASN AIH Tyr Lys AsN Pru Lys t e u Thr Ad9 Met 1, cu Thr Mae
Lys ?he Tyr lief PPõo Lys Lys Alta ihr= Glu Lebu Lys lUs Lcu
GIN Cy; Leu Glu Glu G.iu Lou L.y Pro Lcu G A u Glu Vaal L.eu
AAsN feu A.1a GIN Ser Lys AN rPhe Hia~ Leu A.tg Pro Ark Asp
Leu 1.1te Sep' AsN 11P AsN Gal IIe 1Val Lcu t;lu Lou Lye G::,y
St. r GIu T1ar Thr f 11e Met Lys Clu Tyr Alt, r.op G lu Tti r Ala
Thr .Ile Vaa.1 Glu Phe Lea AIIN Ar+;I Trip ILe TI)r Plat Cyc GINN
See' llu I I e Ser The Leu Tfir
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide comprising a sequence
Pro Thr Ser Ser Sex Thr Lys Lys Thr GIN Leu C1N LOU Glu His LOU Lou Leu Asp
Lou GIN Met Ile Lou ASN G1y Ile Ash AsN Tyr Lys AON pro Lys LOU Thr Arch Piet
Leu Thr Phe Lys Phe Tyr met Pro Lys Lys Ala Thr Glu Leu Lys 1i is Lou GiN Cys
Leu Glu Glu Glu Leu Lys Pro Leo Glu Glu Val Lou ASN Lou Ala GIN Ser Lys ?sN
Who His LOU Arg Pro Arg Asp LOU Ile Ser AsN Ile ASN Val Ile Val Lou Glu Lou
Lys Gly Ser Gl.u Tht Thr Who Met Cys Glu Tyr Ala Asp Glu Thr Ala Tht Ile Val
(lu Phe Leu AsK Arg Trp Ile Thr Phe Cys GIN Set Ile lie Ser Thr Leu 'hr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting essentially of a
sequence
- 2p -
13 41 633
Met--Tyr-A.xg--Met-Gin--Leu-Leu-Ser--Cys -I 1e-Ala-Leu-Ser-
Leu-Ala-Leu-Val-Thr-Asn--=Ser-Ala -Pro-Thx:-Ser-Ser--Ser-
Thr--Lys -Lys-Thr-Gln--Leu--G1n--Leu-GIu-His-Leu-Leuw-Leu-
Asp-Leu-Gln--Me .-ile-LeuR-ASn-Gly-lle--Asn-Asa-Tyr -Lys-
AS n-Pro-Lys -Leu--Thr-Arg-Met--Leu-Thx --Phe--Lys -Phe-Tyr-
Met-Pro- Lys-Lys-Ala-Thr-Glu-Leu-Lys-H .s--Leu--Gln-Cys-
Leu-Glu---G1u--G1u-Leu--Lys-Pro-Leu-G1u-G1u--Va1-Leu-Asn-
Leu--A1a=-G1n--Ser-Lys-Asn-Phe-His-Leu-hrg-Pro-Arg-Asp-
Leu--Ile-Ser-ASn-i1e-Asn-Va1-Ile-Va1-Leu--Glu--lieu--Lys-
Gl.y-Ser. --Glu-Thr -Thr -Phe-rMet-Cys-Glu--TyrW-Al.a-Asp-Glu-
Thr-Ala-Thr-i1e-Val-Glu-Phe-Leu--Asn-Arg--Trp-Ile-Tlr
Phe--Cys-G1n-Ser-Ile-I1e-Ser-Thr-Leu-Thr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting essentially of a
sequence
X--Thr--S6r-Se.r-Sea. -Thr-Lys-Lys-Thr--G1 n-Leu-G1n--Leu-Glu-
H is-Leu--Leu-Leu-Asp-Leu-Gln-Met-I 1e-Leu-A sn-G1y-Ile-
Asn-Asn-Tyr--Lys--Asn-Pro--Lys-L,eu-' hr-Arg-Met-LtiU-Thr-
Phe-Lysr-Phe-Tyr-Met--Pro-Lys -Lys-AIa--Thr-G1u--Leu-Lys-
His -Leu-Gln-Cys-Leu-G1u-G1u-Glu-Leu-YLys-Pro--Leu-Glu--
G1u-Val-Leu-Ain-Leu-Ala-Gl.n-Ser-Lys -Asn-Phe-His-Leu-
A.rg-Pro-Arg--Asp-Leu-I1e-Ser--Asn- iie--Asn--Va1-Tl,e--Va1-
Leu-Giu-Leu-Lys-Gly-Ser-Giu-Thr-Thr--Phe-lief-Cys-Clu-
Tyr-Ala--Asp-G1u-Thr-A1a-Thr-Ile-Vtel.-G, u-Phe-Leu~Asn-
Arg--Trp-I l.e-Thr--Phe--Cys-Gln-Ser-Ile-I1e--Ser-Thr--Leu-
ThrI
wherein X represents Ala-Pro, Pro, Met-Ala-Pro or Met-Pro.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
2q -
1341633
activity, said polypeptide consisting essentially of a
sequence
X-Ala-Pro-Thr-Sc:r-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gin-Leu-Gtu-His-Leu-i-eu-
Leu-A4p-
Leu-Gln-Met-lle-Leu-lsn=,Gly-Il e-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-Thr-Arg-Met-
Leu-
Thr-Phe-Lys-Phe-Tyr-Met-Pro- Lys-Lys-Ala-Thr-Glu-Leu-Lys-Nis-Leu-GIn-Cys-Leu-
Giu,
Glu-Giu-Leu-Lys-Pro- Leu-Giu-Giu-Val-Leu-Asn-Leu-Ala-Glaser-Lys-Asn-Pile-Nis-
Lou-
Arg-Pro-Arg-Asp-Leu-lIe-Ser-Asndle-Asn-Val-1le-Val-Leu-GIu-Leu-Lys-Gly-Ser-Glu-
Thr Thr-Phe-Met-Cys-Giu-Tyi--Ala-Asp-Glu-Thr-Ala-Thr-Ile-Val-Giu-Phe-Leu-Asn-
Arg-
Trp-Ile-Tlir-Phe-Cys-Gin-Ser-lle-tie-Ser-Thr-Leu-Thr,
wherein X is met or H.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting essentially of the
sequence shown in Figure 2.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting essentially of the
following sequence
- 2r -
1341633
Amino kc o c"qu ce i
Ala Pry Thr Sur Sep cae; -Thr Lys Lys Thr GIN Leu GIN L.eu
Glu H s L-, u L e u L.eu Asp Le.u GIN (4ct Iii .L cu A s N Daly I I e
AsN , 5N Tvt Lys AsN fro Lys u Ilir Arq MeIL 1. cu `Ll1r ,, fie
Lys iPhc Tyr IieE Pro Ly z Lys Alit T hl= Glu Lc , Lys His Lcu
G].N Cy:; Lets Glu Glu G,LLi L.eu L.ya Pro Liu G I u G.lu Vol Let,
AsN Lev AIa GIN Ser Lys Aslq +PhL~ Nis Lit u A.rg Pro Arc,, Ais1.
L_eu I e Sett A,h; I it, AsW Vol 1Ic VuI L cu (Iu Leu 1 ys G~' y
Sri Glu i~~r ` h r !'hc titt (Lys C A u Tyr AI:i Asp G:lu Thr Ala
Thr IIe Vai Glu Phe Leu Fuld Arty Trp Ilr Tlii= Flip- Cys GIN
So r l1 I I e 5e: Lhr Liu Thr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting essentially of a
sequence
Pro Thr Set Set set The Lys Lys The GIN Lou GIN Lou Glu His Lou Lou Lou ASP
LOU C;lt filet III I..IU ASH Gly Ile ASH ASH Tyr Lys ASH Pro LYS Lou The Arg
Met
Lelt The Phe Lys Phe Tyr Met Pro Lys Lys Ala 'Iht Glu Leu Lys tl15 Lou G1N Cys
Lou G1u Glv Glu Lou ys pro Lou Glu Glu Val Lou AsN Lou Ala GIN Set Lys AsN
Phe his Lou Aeg Pro Aeg Asp Lou Ile See Ash Tle aASN Val Ile Val LOU Glu Lou
Lys Gly See Glu Thr The Phe Met Cy-s GIu Tyr Ala ASP Glu The Al The Ile Val
Glu Phe Lou Ash Arq Tep ILe The Phe Cys GICM Set Ile lie Scar The Leu The
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of a sequence
2s -
1341633
14e .--T *r--Arg4- at-G n-=Leu--Leu--Ser -Cys-lie-Ala-Leu-Ser-
Leu-A1a-Leu-Vai,-Thhr- Asn-Ser-A1a-pro-Thr-Ser.--Ser-Ser-
Thr-Ly's-LyS-Thr-Oln-:Leu-G .n--Leu-G .uw-His-Leu-Leu-Leu-
Asp-Leu-Gin-Met4-Ile--Leu-~Asn -Gly-Ile-Asn--As -Tyr-Lys
Asn-Pro-Lys-wLeu-` hr-,Arg--Met-Leu-Thr-Phc-Lys--Phe-Tyr--
Met-pro-Lys-Lys--Ala--Thr-G .u-Leu-Lys-Hi.s - aeu-G .n-Cy,--
Leu-G1u*-G1u- Glu-Lr- u-Lys-'ro-Leu-G .u-Gl u-- `al-Lea-ASn-
Leu--Ala-G1n-Ser-Lys-Asn-Phe-His -Leu-hhrq-Pr o--A2: g-Asp-
Leu-Ile-Ser-Asn-1le-Asil--Val.- le- Val-Leu-G .u-Iieu-L,ys-
G1y--Ser--Glu-Thr--Thr. Plte-Met.-CyS-Glu-Tyr-A1a-Asp-G1u-
Thr-Ala-Thr-ile--V l-Glu-Phe--Leu-Asst-Arg-Trp--lle--TUr-
Phe-Cys-G1n- Ser-Ile-Iie-Ser-Thr-Lein-Thr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of a sequence
X-'rhr-SLr-Ser-Ser-Thr -Lys-Lys-Thr-Gln-Leu-Gin--Leu-Glu-
His-~Leu-Leu-Leu-Asp-~Leu-Gl ri-Met-Iie-Lett-Asn--Gly-lle--
Asn-Asn-Tyr-Lys-l%sn-Pro-Lys-Leu-Thr--Arg-Met-Leu-Thr--
Phe-Lys-Phe-Tyr-Met-Pr --Lys-Lys-Ai.a-Thr--G1u-Leu--+Ly;j-
His-Lein-Gln-Cys--Leu-GIu-G .u--Glu-Leu-Tsys-Pro- ,eu--G1u`
Glu-Val_-Leu-.Asn- Leu-.Ala--G1n-Ser--Lys-Asn-Phe--Bis-Leu-
Arg-Pro-Arg-Asp-Leu-lie-Ser-Asn-lle-Asn--Val.--ile-Val
Leu-G1u -Leu--Lys-Gly-Ser-G1u- lthr-Thr--Phe-Met--Cys--Giu-
. Tyr--A1a-Asp-Gi.u-hr-Ala-Thr. -I1e-Va1-G .u-Phe--Leu-Asn-
Arg-Trp-1le-Thr-Phe--Cys --G1n-Sex-I1e-Ile-Sep:--Thr-Leu-
Thr,
wherein x represents Ala-Pro, Pro, Met-Ala-Pro or Met-Pro.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of a sequence
- 2t -
1341633
X-Ala-Pro-Thr-Ser-Ser-Ser-T it-Lys-Lys-Thr-Girl-L u~-Gln-i-eu-Glu-His-Leu-Leu-
Leu-Asp-
Leu-Gin-Met-Ile-Leu-Asn-Gly-lle-Asn-Asn-Tyr-Lys-Asn-Pro-L,ys-Leu-Thr-Arg-Met-
Leu-
Thr-Phe-Lys-Pha-Tyr-feet-Pro-L.ys-Lys-Ala-Thr-Glu-Leu-lys-His-Leu-GIn-Cys-Leu-
Glu-
Giu-Giu==Leu-Lys-pro-L-eu-Glu-Glu-Val-Leu-Asn-Leu-Ala-Gin-Ser-Lys-Asn-Pate-His-
Leu-
Arg-Pro-Arg-Asp-Lou-Ile-Ser-Asn-fie-Asn-Val-lie-Val-Leu-Glu-Leu-L.ys-Gly-Ser-
Glu-
'f'hr- 'hr-Phe-Met-Cys-Glu-Tyr-Ala-Asp-Glu-Thr-Ala-Thr-lia-Val-Glu-Phe-Leu-Asn-
Arg-
Trp-lice-'hr-Phe-Cys-Glh-Ser-lle-Ile-Ser Tlir-Leu-`t'hr,
wherein X is met or H.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of the sequence shown
in Figure 2.
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of the following
sequence
Acii Acja quenc i
1 h 7o Tl~r SLr SeR Se !LYS Thr GIN Lau G1tt Lt~u
Glu Flit Leu Leu Lr.u Asp Liu GIN i1ct I1n 'Lau As1t Gly Ile
Asst AsN Tyr Lys AsN Pro Lys 1.eu Thr Ark t,Iav 1.eo Thhi file
Lys the Tyr lies: Pro Lys Lys Mu 'I"i`hr Gl.u Le;., Lyo His Lcu
{;]14 Cyr; L e u Clu Gku G.lu Leu t,y-a Pro Ltu Glu Glu Vol Leu
ltstt Leu Ala GIN Ser Lys AsH P h a H.i;s Ltti A.rg Pro Arn Asir
Leu Ile Ser AsN II A - H U a.'1 tic Vol Lcv Giu Leu Iycs G?y
5, x G1i The Ttt.r Pte Met Gys C1u Tyr. Alm Asp Glu Thi Ala
Thr Ile Va.I Glu Phhe Leo AsH Ax Trp 12.e Thr PIIe C ys GIN
Ser lie lie Ser= Thr Leu Thr
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity, said polypeptide consisting of a sequence
- 2u -
1341633
Pro Vhr Ser Ser Ser Thr Lys Lys Thr GIN Lou GIN Lau Glu His Lou Lou Lou Asp
E.eu Glti 1iet Ile Lou AsN Gly Ile Ash ASH Tyr Lys ASH Pro Lys Lou Thr .erg
Mat
Lou Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr iglu Leu Lys His Lou GiN
C_y.~s
Leu (-, lu Glu Glu Leu Lys Pro Leu cllu Glu Val Leu AsN LOU Ala GiN Ser Lys r
sN
Phe His Lou Arg Pro Arch Asp Lou Ixe Ser ASH Ile ASH Val Ile Val Lou Glu Leu
Lys G I y Scar G I u Thr. Thr Phe Met Cys Gi,u Tyr,' Ala Asp Clu Thr Ala Thr
tie V11
G u Phe Lou AsH Arg Tr'p tie Thr Phe Cys GIN Ser Ile Ile Sew Thr L uz '".'h
In accordance with the invention, there is
provided an isolated polypeptide having human interleukin-2
activity which consists of a 132 amino acid sequence, said
sequence containing Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-
Leu-Gln-Leu-Glu-His-Leu-Leu-Leu-Asp-Leu in N-terminal and
Thr as the C-terminal amino acid.
In accordance with the invention, there is
provided an isolated polypeptide as described herein,
wherein said sequence further contains at least one of the
following fragments:
1) Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-
Glu;
2) His-Leu-Leu-Leu-Asp-Leu-Gln-Met;
3) Ile-Leu-Asn-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-
Thr-Arg-Met;
4) Leu-Thr-Phe-Lys-Phe-Tyr-Met;
5) Leu-Lys-His-Leu-Gln-Cys-Leu-Glu-Glu-Glu;
6) Glu-Glu-Leu-Lys-Pro-Leu-Glu or Glu-Leu-Lys-Pro-Leu-Glu-
Glu;
7) Glu-Leu-Lys-Pro-Leu-Glu or Leu-Lys-Pro-Leu-Glu-Glu;
2v -
17 41633
8) Val-Leu-Asn-Leu-Ala-Gln-Ser-Lys-Asn-Phe-His-Leu-Arg-
Pro-Arg-Asp-Leu-Ile-Ser-Asn-Ile-Asn-Val-Ile-Val-Leu-
Glu; and
9) Thr-Ala-Thr-Ile-Val-Glu.
In accordance with the invention, there is
provided an isolated polypeptide as described, wherein said
sequence further contains the following fragments:
1) Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr-Gln-Leu-Gln-Leu-
Glu;
2) His-Leu-Leu-Leu-Asp-Leu-Gln-Met;
3) Ile-Leu-Asn-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu-
Thr-Arg-Met;
4) Leu-Thr-Phe-Lys-Phe-Tyr-Met;
5) Leu-Lys-His-Leu-Gln-Cys-Leu-Glu-Glu-Glu;
6) Glu-Glu-Leu-Lys-Pro-Leu-Glu or Glu-Leu-Lys-Pro-Leu-Glu-
Glu;
7) Glu-Leu-Lys-Pro-Leu-Glu or Leu-Lys-Pro-Leu-Glu-Glu;
8) Val-Leu-Asn-Leu-Ala-Gln-Ser-Lys-Asn-Phe-His-Leu-Arg-
Pro-Arg-Asp-Leu-Ile-Ser-Asn-Ile-Asn-Val-Ile-Val-Leu-
Glu; and
9) Thr-Ala-Thr-Ile-Val-Glu.
In accordance with the invention, there is
provided a pharmaceutical composition, comprising the
isolated polypeptide described herein in association with a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
- 2w -
1 3 4 1 633
Figure 1 shows a restriction endonuclease cleavage
map of a cloned gene coding for IL-2 polypeptide.
Figure 2 shows the base sequence of the cloned
gene.
Figure 3 shows the plasmid vector pTrS-3.
Figure 4 is a flow chart showing the construction
of recombinant DNA pTIL2-21.
Figure 5 is a flow chart showing the construction
of a recombinant DNA pTIL2-22.
In the Figures, "A", "G", "C" and "T" represent
deoxyadenylic acid, deoxyguanylic acid, deoxycytidylic acid
and thymidylic acid, respectively.
- 2x -
1341633
BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT
The new IL-2 polypeptides of the present invention
bear threonine as the C-terminal amino acid and no sugar
moiety. One example of the IL-2 polypeptides has alanine
as the N-terminal amino acid, and more precisely has the
following amino acid sequence I.
Amino Acid Sequence I
Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr G1N Leu GIN Leu
Glu His Leu Leu Leu Asp Leu GIN Met Ile Leu AsN Gly Ile
AsN AsN Tyr Lys AsN Pro Lys Leu Thr Arg Met Leu Thr Phe
Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu
GIN Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu
AsN Leu Ala GIN Ser Lys AsN Phe His Leu Arg Pro Arg Asp
Leu Ile Ser AsN Ile AsN Val Ile Val Leu Glu Leu Lys Gly
Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala
Thr Ile Val Glu Phe Leu AsN Arg Trp Ile Thr Phe Cys GIN
Ser Ile Ile Ser Thr Leu Thr
Another example of the IL-2 polypeptides has
proline as the N-terminal amino acid, and more precisely
has the following amino acid sequence II.
Amino Acid Sequence II
Pro Thr Ser Ser Ser Thr Lys Lys Thr GlN Leu GIN Leu Glu
His Leu Leu Leu Asp Leu GIN Met Ile Leu AsN Gly Ile AsN
AsN Tyr Lys AsN Pro Lys Leu Thr Arg Met Leu Thr Phe Lys
Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu GIN
Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu AsN
Leu Ala GIN Ser Lys AsN Phe His Leu Arg Pro Arg Asp Leu
3 -
1341633
Ile Ser AsN Ile AsN Val Ile Val Leu Glu Leu Lys Gly Ser
Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr
Ile Val Glu Phe Leu AsN Arg Trp Ile Thr Phe Cys GIN Ser
Ile Ile Ser Thr Leu Thr
The IL-2 polypeptides of the present invention
were produced in cells of Escherichia coil constructed by
gene-recombination technique, by culturing the cells in
a nutrient medium. Construction of Escherichia .coli
capable of producing the IL-2 polypeptides of the present
invention was performed by the manner shown in Examples
1 and 2.
The IL-2 produced intracellularly or extra-
cellularly is recovered by any known method, such as
precipitation with ammonium sulfate, dialysis to remove
salts (under normal or vacuum pressure), gel filtration,
chromatography, preparative flat-bed iso-electric focusing,
gel electrophoresis, high performance liquid chromatography
(hereinafter "HPLC"), (ion exchange, gel filtration and
reverse phase chromatography), and affinity chromatography
TM
on dye bound carrier, on activated Sepharose 4B coupled
with monoclonal antibody against said IL-2 or on lectin
bound Sepharose 4B and the like. Methods of recovery, and
purification of IL-2, are described in Watson et al., J.
Exp. Med., 150, 849-861 (1979), Gillis et al., J. Immunol.,
124, 1954-1962, (1980), Mochizuki at al., J. Immunol.
Methods 39, 185-201, (1980), and Welte, K. at al., J. Exp.
Med., 156, 454-464 (1982).
4 -
1 3 4 1 633
The activity of IL-2 may be ascertained by the
microassay procedure principally discussed by Gillis et al.
(Gillis, S. et al., J. Immunol., 120, 2027-2033 (1978)).
The assay monitors the IL-2 dependent cellular proliferation
of a cytotoxic T lymphocyte cell lines (hereinafter "CTLL")
generated according to the methods described by Gillis
et al., that is, 4 x 103 CTLL cells are inoculated into
100 p.Q of RPMl 1640 medium containing 2% FCS in 96 well
flat-bottomed microplates together with 100 p.¾ of the serially
diluted translation products. After 20 hours incubation
at 37 C in 5% CO2 incubator, cells are pulsed for 4 hours
with 0.5 pCi of 3H-TdR, harvested onto glass fibre strips
with the aid of an automated cell harvester and then the
incorporated radioactivity is measured by liquid scintil-
lation counting. By these assay procedures, the CTLL cells
cultured in the presence of IL-2 were found to incorporate
3H-TdR in a dose dependent manner resulting in the definite
calculation of the amount of IL-2 contained in test samples.
IL-2 possesses the activity to promote the prolif-
eration of T lymphocytes, which enables the measurement
of IL-2 activity using an index of T cell growth activity.
That is, five CTLL cells are transferred into 100 pi of
DMEM containing 2% FCS in 96 well flat-bottomed microplates
together with 100 pi of the serially diluted translation
products. After 72 to 96 hours incubation at 37 C in a
5% CO 2 incubator, the number of cells grown and activated
is counted under microscopy. As a positive external control
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13 41 633
group, 100 units/ml, 10 units/ml of IL-2 are added and the
IL-2 activity of the test sample is calculated in comparison
with the number of grown viable cells in these control
groups.
The polypeptide thus obtained shows the same
biochemical and biological behavior as has been known for
IL-2 produced by mammalian cells by mitogen stimulation,
and has IL-2 activity. The molecular weight is around
15,000- dalton and IL-2 activity was completely neutralized
or precipitated with monoclonal anti-IL-2 antibody in the
Tm
presence or absence of immunoadsorbents, such as Igsorb
(Enzyme Center). In immunoelectrophoresis, the IL-2
polypeptide shows only a single precipitate against the
corresponding anti-IL-2 antibody. The IL-2 activity remains
stable after reduction with 2-mercaptoethanol, and is
resistant to treatment with DNAse and RNAse as well as to
heat treatment at 56 C for 30 min. The activity is stable
at a pH between pH 2 to 9. The IL-2 produced could promote
the growth of monoclonal functional T cells (cytotoxic T
lymphocyte), enhance the thymocyte mitogenesis, give rise
to the generation of anti-tumor specific cytotoxic T
lymphocytes from memory state in the absence of the antigen,
and could be used to augment natural killer cell activity
against YAC-1 and RLtl cells.
The IL-2 polypeptide preparations of the present
invention are free from physiologically active substance
produced by human cell, and can be more convenient for the
6 -
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therapeutic use than the known IL-2 polypeptide preparation
produced by a human cell.
Example 1
(1) Human T leukemia cell line, Jurkat cells
(freely available in Japan, W. Germany and United States)
were suspended in RPMI 1640 medium containing 10 vol/vol %
FCS and were irradiated with X-ray till 10,000 roentgen
at a room temperature for 50 seconds using X-ray irradiation
apparatus Exs 150/300 - 4 (Toshiba, Japan), and thereafter
the irradiated cell was cultured for 5 days at 37 C in 5%
CO2 incubator at an initial cell density of 1 x 105
cells/ml in the culture medium mentioned above. The mutated
cells (0.2 cells/well) were placed in wells 10 pieces of
flat-bottomed microplates having 96 wells, and cultured
at 37 C in 5% CO2 incubator for 21 days.
Clones obtained from the wells showing growth
were repeatedly transferred into fresh culture medium to
propagate the clone sizes, and the propagated clones were
cultured for 24 hours at an initial cell density of 1 x 106
cells/ml in the presence of 50 pg/ml of Con. A and IL-2
activity was measured according to the methods aforementioned.
Consequently a human T cell line designated as Jurkat-111
(hereinafter "J-111") (ATCC CRL8129), cloned from parent
Jurkat, was selected, of which productivity of IL-2 was
increased 40 times as much as that of the parent strain.
The cloned cell line J-111 could grow under conventional
conditions and the growth rate shows almost the same with
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ordinary Jurkat cells.
(2) Cells (1 x 105/ml) of J-111 were inoculated
in 1,000 ml of serum free synthetic culture medium RITC
55-9 (Sato, T. et al., Exp. Cell Res., 138, 127-134,
(1982)) in roller culture bottles (Falcon 3027) and cultured
for 4 days at 37 C, and cells propagated were harvested by
centrifugation. The harvested cells were again inoculated
in the medium mentioned above which had been added with
25 pg/ml of Con. A to contain 4 x 106 cells/ml. In four
batches of roller culture bottles (Falcon), 1,000 ml of
the inoculated culture medium was placed into each batch.
The cultivation was continued for 6 hours with rotating.
Jurkat cells (1.2 x 106) thus stimulated with
25 ig/ml of Con. A for 6 hours were suspended in 8,000 ml
of phosphate buffer balanced with saline (hereinafter "PBS").
The cells were washed twice by centrifugation and were
resuspended in 800 ml of RSB solution (10 mM Tris-HC1, pH
7.5, 10 mM NaCl, 1.5 mM MgC12) containing Ribonucleosides-
Vanadyl Complex (10 mM), an inhibitor of nuclease. Then
a detergent NP-40 was added to contain 0.05 6 as final
concentration, followed by gentle mixing and the cell nuclei
were removed by centrifugation for five minutes at 3,000 rpm
at 4 C. SDS (0.5%) and EDTA (5 mM) were added to the
supernatant and cytoplasmic RNA was extracted by addition
of equal volume of phenol. After three times extraction
with phenol, RNA was precipitated with two times volume
of ethanol and precipitates were collected by centrifugation,
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which were solubilized in 10 mM Tris-HC1 of pH 7.5. The
amount of RNA obtained was 196 mg.
Fractionation of mRNA was carried out using
affinity chromatography on oligo (dT)-Cellulose (P. L.
Biochemicals, Type 7). An adsorption solution was a solution
of pH 7.5 containing 20 mM Tris-HC1, 0.5 M NaCl, 1 mM EDTA
and 0.5 0' SDS and elution was carried out with water and
mM Tris-HC1 (pH 7.5) by turns after washing the column
with the buffer (20 mM Tris-HC1, pH 7.5, 0.5 M NaCl, 1 mM
EDTA). The resultant mRNA eluted was 3.6 mg. Next, 2.4 mg
of the mRNA obtained was fractionated by sucrose density
gradient centrifugation (5 to 2.5 0' sucrose density gradient
in a solution of pH 7.5 containing 50 mM Tris-HC1, 1 mM
EDTA and 0.2 M NaCl, centrifuged at 26,000 rpm for 24 hours
at 4 C, and 11 to 12S fraction of mRNA was fractionated into
fractions No. 12, 13, 14 in the amount of 50 jig, 46 pg and
60 pg, respectively.
(3) The mRNA obtained in fraction No. 13 was
microinjected into the oocyte of Xenopus laevis (50 ng
mRNA/egg) and the culture supernatant was served for the
assay of IL-2 activity. As shown in Table 1, the increase
of the incorporation of 3H-TdR and the increase of number
of activated T lymphocytes were confirmed, clearly verifying
that mRNA in this fraction contains human IL-2 mRNA.
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Table 1
(a)
Sample Dilution Uptake of 3H-TdR Amount of IL-2*
(cpm) (unit/ml)
Control I - 553 0
(Medium for assay)
Control II x 2 590 0
(Supernatant of egg x 32 572
culture non-treated)
Translation product x 8 14,683 32
of fraction 13
x 32 10,165
(b)
Cell number of Amount of IL-2*
Dilution T-lymphocyte
(No./well) (unit/ml)
Control I x 2 0 0
(Medium for assay) x 16 0
Control II x 2 0 0
(Supernatant of egg x 16 0
culture non-treated)
Translation product x 2 115 40
of fraction 13
x 16 55
* The unit was calculated by comparing the amount of
incorporated 3H-TdR with that of standard IL-2 (10
unit/ml) according to probit analysis.
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(4) Thereafter cDNA was synthesized in vitro
from No. 13 fraction of 11 to 125 mRNA containing IL-2 mRNA
and recombinant DNA was constructed with the plasmid vector
pBR 322. With the recombinant DNA, Escherichia coli was
transformed, and clone acquired IL-2 cDNA clones was
selected, as follows:
Fifty mM Tris-HC1 buffer (pH 7.5), 30 mM NaCl,
6 mM MgCl2, 5 mM dithiothreitol (hereinafter "DTT"), 0.5 mM
of each dATP, dGTP, dCTP, dTTP_(dCTP contained 32P radio-
labelled one), 0.7 pg oligo (dT)10, 10 pg mRNA and 15 unit
AMU reverse transcriptidase (J. W. Beard) were mixed and
maintained for 90 min. at 411C.
After termination of the reaction, DNA was
recovered as ethanol precipitates after the phenol
treatment, and DNA was solubilized in a solution of pH 7.5
containing 20 mM Tris and 1 mM EDTA.
Two point five pg of ss-cDNA was synthesized.
To remove mRNA present in this solution, the solution was
made 0.33 N-NaOH by addition of NaOH, allowed to stand for
15 hours at a room temperature, then the solution was
neutralized with equal volume of 1 M-Tris-HC1 of pH 7.5
TM
and passed through "Sephadex G-50" column. The recovered
cDNA was 1.8 11g.
Fifty mM phosphate buffer (pH 7.5), 10 mM MgC121
mM DTT, 0.75 mM of each dATP, dGTP, dCTP, dTTP (dCTP
contains 3H radiolabelled one), 1.8 Vg ss-cDNA, and 8 unit
of polymerase I (BRL, United States) were mixed and were
11
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allowed to react for 15 hrs. at 15 C. After the termination
of the reaction, DNA was recovered as ethanol precipitate,
after treatments with phenol and with chloroform. 1.10 ug
of ds-cDNA was generated. A mixture of 50 mM sodium acetate
(pH 4.5), 0.2 M NaCl, 1 mM ZnCl2 and 1.10 pg of ds-cDNA
was incubated for 20 min. at 37 C, added with 0.25 unit
of nuclease Sl (Sankyo, Japan), and incubated further for
15 min.
After the termination of the reaction, the reaction
product treated twice with phenol was applied onto Sephadex
G-50 to get 0.55 pg of ds-cDNA.
A mixture of 0.14 M potassium cacodylate, 30 mM
Tris base, 0.1 mM DTT, 1 mM 00012, 0.64 mM 32P-dCTP (spc.
act. 2.7 x 106 cpm/n mol), 0.55 pg of ds-cDNA and 5 unit
of terminal transferase (BRL) were incubated for 7 min.
at 37 C, then applied onto Sephadex G-50 column after phenol
treatment to get 0.50 jig DNA as ethanol precipitates. The
recovered DNA was found to be extended with around 50 dCMP
residues at the both 3' terminus.
Ten pg of pBR 322 DNA was cleaved with restriction
enzyme PstI, and 3'-termini of the cleaved DNA were added
with dGMP chain, by the same method as that used in the
addition of dCMP to ds-cDNA mentioned above, except dGTP
was used in place of dCTP.
(5) A mixture of 50 mM Tris-HC1 (pH 7.5), 0.1 M
NaCl, 5 mM EDTA, 0.05 jig of pBR 322 elongated with dGMP
residues and 0.01 pg of cDNA extended with dCMP was incubated
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firstly for 2 min. at 65 C, then for 120 min. at 46 C, for
60 min. at 37 C and finally for 60 min. at a room temperature.
E. coli X 1776 (Curtiss III, R. et al., in Molecular Cloning
of Recombinant DNA, (W. A. Scott & R. Werner ed.) Academic
Press, (1977)) was inoculated in 50 ml of L broth containing
100 pg/ml of diaminopimelic acid, 50 pg/ml of thymidine,
1 0' tryptophan, 0.5 0' yeast extract, 0.5 0' NaCl and 0.1 0' glucose
and cultured in shaking at 37 C until the absorbance of
culture liquid at 562 nm became around O.D 0.3. After
the termination of the culture, the culture liquid was left
at 0 C for 30 min., then the bacterial cells were collected
by centrifugation followed by twice washing with 25 ml of a
solution containing 5 mM Tris-HC1 (pH 7.6), 0.1 M NaCl,
mM MgCl2 and 10 mM RbCl.
Cells thus obtained were suspended in 20 ml of
a solution containing 5 mM Tris-HC1 (pH 7.6), 0.25 M KC1,
5 mM MgC12, 0.1 M CaCl 2 and 10 mM RbCl and were left at
0 C for 25 min., then cells were collected to resuspend
them into 1 ml of the same solution, the recombinant DNA
described above was added into 0.2 ml of the cell suspension
and the suspension was left at 0 C for 60 min. Then 0.7 ml
of L broth was added to culture in shaking for 30 min. at
37 C. Thus obtained culture medium (0.1 ml) was thoroughly
spread on the surface of 1.5 0' agarose medium composed of
L broth containing 100 pg/ml diaminopimelic acid, 50 pg/ml
thymidine and 15 pg/ml tetracycline, and incubated at 37 C
for two days.
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Four hundred and thirty two colonies appeared
were divided into 18 groups, each containing 24 different
bacterial clones, inoculated in 200 ml of L-broth containing
100 pg/ml of diaminopimelic acid, 50 pg/ml of thymidine
and 10 Mpg/ml of tetracycline and cultured in shaking at
37 C for 5 to 7 hrs. Then, 200 ml of fresh L-broth contain-
ing chloramphenicol at a final concentration of 170 pg/ml
was added to culture further for an overnight. Thus
amplified plasmid DNA was purified according to a con-
ventional mean. Clones possessing IL-2 cDNA were screened
by a mRNA hybridization-translation assay (hereinafter "H-T
assay"). H-T assay here employed was carried out as
follows: Purified DNA (25 pg) was cleaved with restriction
enzyme Hind III, treated with phenol three times, treated
with phenol-chloroform and with chloroform, respectively,
precipitated with ethanol, washed with 80 6 ethanol and
dissolved in 40 p of 8096 formamide. The reaction mixture
was heated for denaturation at 90 C for 5 min., then diluted
to 1.3 ml with 10 x SSC (1.5 M NaCl, 0.15 M sodium citrate).
The DNA was thereafter fixed onto nitrocellulose filters,
which filters were dried up at 80 C for 3 hrs. and incubated
for 18 hrs. at 37 C in the solution containing 50% formamide,
20 mM Pipes of pH 6.5, 0.75 M NaCl, 5 mM EDTA, 0.2 6 SDS
and 250 pg of poly (A) mRNA from induced J-111 cells to
hybridize the DNA fixed on filters with IL-2 mRNA. Then
the filters were washed at 65 C three times with solution
consisting of 10 mM Pipes of pH 6.5, 0.15 M NaCl, 1 mM Pipes,
14 -
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mM NaCl solution and treated with 0.5 mM EDTA, 0.1 6 SDS
solution at 95 C for 1 min. to recover the hybridized mRNA
from the filters. Thus extracted mRNA was purified on oligo
dT-Cellulose column according to the conventional methods
and injected into Xenopus oocytes to determine the IL-2
activity of translated proteins. One out of 18 groups,
each consisting of 24 clones, gave positive 48 unit/ml IL-2
activity in 3H-TdR incorporation assay described previously,
while others being clearly negative. Then 24 single colonies
belonging to the positive group were inoculated in 200 ml of
L-broth possessing the same composition described, cultured
aerobically for 5 to 7 hrs. at 37 C and similarly chloram-
phenicol containing fresh L-broth was further added. After
amplification of plasmid DNA by an overnight culture, plasmid
DNA was similarly purified according to the standard pro-
cedures. After cleavage of about 5 pg of each plasmid DNA
with Hind III, each plasmid DNA was bound to nitrocellulose
filters similarly. The filters were hybridized with IL-2
mRNA and hybridized mRNA was recovered to inject into
Xenopus oocyte to determine the IL-2 activity of translated
proteins. As shown in Table 2, only plasmid DNA purified
from a single colony, designated as p3-16, gave the positive
IL-2 activity. Therefore this clone was identified as the
clone possessing IL-2 cDNA (E. coli X 1776/p3-16 AJ 11995
(FERM-BP-225)). Thus plasmid DNA, p3-16, was confirmed to
share exactly the DNA (IL-2 gene) capable of forming the
specific hybrid with IL-2 mRNA.
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Table 2
(a)
Sample Dilution Uptake of 3H-TdR Amount of IL-2
(cpm) (unit/ml)
Control I - 2,010 0
(Medium for assay)
Control II x 2 2,120
(Supernatant of x 32 2,482 0
culture liquid of
non-treated egg)
Translation product x 2 20,453 58
of mRNA
x 32 20,961
(b)
Cell number of Amount of IL-2
Sample Dilution T lymphocyte
(cells/well) (unit/ml)
Control I - 0 0
(Medium for assay)
Control II x 2 0 0
(Supernatant of x 32
culture liquid of
non-treated egg)
Translation product x 2 88 32
of mRNA*
x 32 42
mRNA hybridized with cDNA from plasmid p3-16.
- 16 -
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(6) The cDNA insert of plasmid p3-16 showed
characteristics to be cleaved by restriction enzyme XbaI
at a single site and by BstNI at two sites, (at upstream
and downstream of XbaI cleavage site). However the plasmid
p3-16 contained a cDNA insert consisting of about 650 base
pairs, which apparently corresponds to a part of IL-2 mRNA
of 11 to 12S size.
Therefore another cDNA library were prepared
according to the procedure of Land et al. (Land et al.,
Nucleic Acids Res., vol 9, p2551, (1981)) using IL-2 mRNA
as a template. Single stranded cDNA (1.6 pg) was synthe-
sized by using 4 pg of IL-2 mRNA elongated by dCMP residues,
and ds-cDNA was synthesized by using oligo (dG) 12-18 as
the primer for DNA polymerase I (Klenow fragment). The
cDNA (0.6 jig) longer than 680-base pair DNA size marker
was obtained by a sucrose gradient centrifugation and
inserted into the PstI site of pBR 322 by the standard G-C
tailing method. After transformation of E. coli Z 1776
by the recombinant DNA, approximately 2,000 colonies were
screened by in situ hybridization method of Grunstein-Hogness
with nick-translated p3-16 cDNA insert as the probe and
the colony containing plasmid pIL 2-50A containing around
850 base pairs and the transformed clone (E. coli X 1776/pIL
2-50A, AJ 11996 (FERM-BP-226)) were identified. A re-
striction endonuclease cleavage maps of the cDNA insert
of pIL 2-50A are shown in Fig. 1. To isolate a gene coding
for IL-2 peptide from transformed E. coli X 1776 pIL 2-50A,
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plasmid DNA was digested with restriction enzyme PstI after
isolation of DNA region from the cells according to the
conventional means. Thus produced smaller fragment .a.raong
generated two DNA fragments was DNA gene coding for IL-2
peptide. The complete nucleotide sequence of the PstI
insert from pIL 2-50A was determined by the procedure of
Maxam and Gilbert (Maxam, A. W. et al., Enzym. 65, 499-560,
1980), and the whole structure is shown in Fig. 2.
(7) A plasmid which should direct the synthesis
of human IL-2 in E. coli cells was constructed as follows.
A plasmid pTIL 2-21 was constructed from pTrS-3 (Nishi T.,
Taniguchi T. et al., SEIKAGAKU 53, 967, (1981)), of which
restriction map is shown in Figure 3, and pIL 2-50A
containing the IL-2 cDNA by the manner illustrated in
Fig. 4. Plasmid pTrS-3 include insertion of the region
of Trp promoter and Shine Dalgarno (hereinafter "SD")
between EcoRI site and Clal site of pBR 322.
Plasmid pTrS-3 (10 pg) was at first cleaved with
the restriction enzyme Sail and the Sall site was rendered
flush by the treatment with DNA polymerase (Klenow fragment)
or with T4 DNA polymerase. After cleavage with C1aI, a
larger fragment, containing the trp promoter region, was
isolated by agarose gel electrophoresis in a conventional
manner to recover 3 pg of DNA.
On the other side, 11 jig of pIL 2-50A insert into
Psti was cleaved with HgiAl, treated with T4 DNA polymerase
and a larger fragment was isolated and purified by agarose
- 18 -
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gel electrophoresis. Thus cDNA fragment coding for 132
amino acids of IL-2 was obtained in an amount of 7.2 11g.
Then 0.45 pg of the fragment containing a trp promoter
(described above), 0.5 pg of HgiAl-PstI fragment containing
IL-2 cDNA and synthetic oligonucleotides (5') CGATAAGC
TATGGCA (3'), and (3') TATTCGATACCGT (5') (each 20 pmole),
both of which were phosphorylated at 5'-terminus, were
ligated with 1.0 unit of T4 DNA ligase in 66 mM Tris-HCl
of pH 7.5 containing 6.6 mM MgC12, 1 mM ATP and 10 mM DTT,
and the mixture was allowed to react at 4 C overnight.
Thus ligated plasmid was then used to transform
E. coli HB101. Among the transformants appeared on L broth
agar plate containing ampicillin, the target transformants
were selected as follows. The candidate transformants able
to hybridize with both of IL-2 cDNA and synthetic oligo-
nucleotides were firstly selected by colony hybridization
method, then the transformants possessing the insertion
of DNA fragment initiating from CCT sequence at position
III to 113 in Fig. 2 (CCTACT ----- ) just downstream of
ATG GCA sequence were selected by PstI, XbaI cleavage.
The E. coli HB101 containing pTIL2-21a or pTIL2-21b was
cultured under the conventional conditions known for the
propagation of microorganisms. The cells were grown in
ml of X broth (2.5 % Bactotrypton, 1.0,10' yeast extracts,
0.1 0' glucose, 20 mM MgSO4, 50 mM Tris-HC1, pH 7.5) contain-
ing 25 pg/ml streptomycin and 25 jig of ampicillin at 37 C
for an overnight. One ml of the culture suspension was
- 19 -
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inoculated into the same/. broth (100 ml) and cultured at
37 C. When O.D at 650 mp arrived around 1.5-2.0, 3-indole
acrylic acid (IAA) was added. Three hours after the
addition of inducer, the cells were collected, washed with
20 mM Tris-HC1 (pH 7.5, 30 mM NaCl) and resuspended into
8 ml of the same buffer. For the efficient functioning
of Trp promoter inducers such as IAA was added at a final
concentration of 50 pg/ml. Thus produced proteins in
bacterial cells were extracted. by sonication (0 C, 2 min.)
or lysozyme (8 py) digestion (0 C, 20 min.) followed with
three successive freeze-thawing. According to this pro-
cedures IL-2 was usually extracted from organisms. The
extracted IL-2 activity ranged from 10,000 to 120,000
units/mi.
Escherichia coli HB101 possessing pTIL2-21a
(AJ12013) and Escherichia coli HB101 possessing pTIL2-21b
(AJ12014) have been deposited in the assession numbers of
FERM-BP248 and FERM-BP249 respectively.
(8) Escherichia coli AJ12013 (FERM-BP248) was
inoculated on 10 1 of an L medium (containing 1%
tryptone, 0.5% yeast extract, 0.5 0' NaC1 and 0.1 0' glucose)
containing 25 pg/ml of ampicilline and 25 pg/ml of
streptomycin and cultured. When optical density at 650 nm
reached about 1.0, 3-indol-acrylic acid was added to the
medium at a concentration of 50 pg/ml and cells were
collected 2 hours after. After washing the cells with a
20 mM Tris buffer (pH 7.5) containing 30 mM NaCl, the cells
20 -
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were again suspended in 180 ml of the same buffer solution.
Then, 20 ml of a lysozyme solution and further 2 ml of 0.5 11
EDTA (pH 8.0) were added to the suspension. Thereafter,
the mixture was allowed to stand for 20 minutes at 0 C.
By subsequently performing freezing-thawing at -50 C and
37 C 3 times, the cells were disrupted. Ultra centrifugation
was carried out at 30,000 rpm for 30 minutes to obtain an
extract of the cells from the disrupted cells.
Out of the extract 160 ml (total protein content
2.4 g, IL-2 activity 3 x 105 u/.ml, specific activity
2 x 104 p/mg) was passed through a column (32 mm diameter x
65 mm) filled up with 50 ml of porous glass beads (CPG-10,
pore size 350 angstrom, 120-200 mesh, manufactured by
Electro-Nucleonics Co., Ltd.) which had been previously
equilibrated with a 0.1 M tris-(hydroxymethyl)aminomethane-
-hydrochloric acid buffer solution of pH 7.7 containing 0.2 M
sodium chloride to adsorb IL-2 thereto. Thereafter, the column
was washed with 100 ml of the aforesaid buffer solution
and then IL-2 was eluted out with 200 ml of a 0.1 M
tris-(hydroxymethyl)aminomethane-hydrochloric acid buffer
solution of pH 7.7 containing 0.75 M potassium thiocydnate.
After dialyzing 150 ml of the obtained IL-2
eluate to a 0.07 H acetic acid-sodium acetate buffer solution
having pH 6.0 for 48 hours, the eluate was passed through a
column (22 mm diameter x 105 mm) filled up with 40 ml of
"CM-Sephadex C-25" (manufactured by Pharmacia Co., Ltd.)
which had been previously equilibrated with the same buffer
solution to adsorb IL-2 thereto. After subsequently washing
21 -
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the column with 100 ml of the same buffer solution, the
adsorbed IL-2 was eluted out with 100 ml of a 0.5 M acetic
acid-sodium acetate buffer solution of pH 6Ø
Solid ammonium sulfate was added to 80 ml of the
obtained eluate to render 80 0' saturation. After settling
overnight, formed precipitates were collected by centrifu-
gation and dissolved in 10 ml of a 0.05 M phosphoric acid-
sodium phosphate buffer solution having pH 7.0 and contain-
ing 1.25 M sodium chloride. Using 500 ml of "Sephadex G-75
Super Fine" (manufactured by Pharmacia Co., Ltd.) equi-
librated with the same buffer solution, gel filtration (32 mm
diameter x 65 cm) was performed. IL-2 was eluted out as
a single active peak at a molecular weight of 14,000 to
16,000 daltons.
Glucose was added to 20 ml of the obtained IL-2
fraction so as to have the final concentration of 1 M, and
the mixture was passed through a column (10 mm diameter x
6 cm) filled up with 5 ml of "Phenyl Sepharose CL-6B"
(manufactured by Pharmacia Co., Ltd.) previously equilibrated
with a 0.05 M phosphoric acid-sodium phosphate buffer
solution having pH 7.0 and containing 1.25 M sodium chloride
and 1 M glucose to adsorb IL-2 thereto. Next, the column
was washed with 15 ml of the same buffer solution. There-
after the adsorbed IL-2 was eluted out with 30 ml of a
0.05 M phosphoric acid-sodium phosphate buffer solution
having pH 7.0 and containing 0.1 M sodium chloride and 1 M
glucose.
22 -
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Using a Hitachi* 638-30 high speed liquid chromato-
graphy apparatus (manufactured by Hitachi Ltd.), 5 ml out
of 20 ml of the obtained IL-2 fraction was passed through
a column (4.6 mm diameter x 75 mm, manufactured by Beckmam
Co., Ltd.) for high speed liquid chromatography filled up
with "Ultrapore*RPSC", which had been previously equi-
librated with a 0.5 M acetic acid-triethylamine buffer
solution of pH 4.0, at a flow rate of 0.5 ml/min. There-
after, elution was performed using the aforesaid buffer
solution (hereafter referred to as Solvent A) and a 80%
v/v 1-propanol aqueous solution (hereafter referred to as
Solvent B).
Solvent A alone was flown for the initial 10
minutes; during 10 to 22 minutes, solvents were flown by
varying from 100 0' Solvent A to 70 0' Solvent A + 30 0' Solvent
B according to the linear gradient method;'and the solvents
were flown down during 22 to 86 minutes by varying from
70 0' Solvent A + 30 0' Solvent B to 30 0' Solvent A + 70 0' Solvent
B according to the linear gradient method. Detection of
proteins was performed by measuring the absorbance at 280 nm
using a wavelength variable ultraviolet absorbance monitor,
Hitachi 638-41 (manufactured by Hitachi Ltd.). Human IL-2
was eluted out as a single peak 70 minutes after from the
initiation of the elution. The recovery rate from the
extract of the bacteria was 30%. The thus obtained IL-2
showed an activity of 5 x 107 units per 1 mg of the protein.
(9) The obtained IL-2 showed a single band at the
* Trade Mark
23 -
1341633
location of a molecular weight of about 16,000 daltons by
SDS-polyacrylamide gel electrophoresis. The analysis of
the N-terminal residue was conducted in a conventional
manner by the dansyl method and as a result, only alanine
was detected as the N-terminal amino acid.
Next, using about 40 pg (250 picomoles) of the
obtained IL-2, the amino acids constituting IL-2 were
sequentially determined from the N-terminal by the automatic
Edman (degradation) method (The Journal of Biological
Chemistry, vol. 256, pages 7990-7997, 1981) using a Gaseous
Phase Protein Sequencer Model 470A (manufactured by Applied
Biosystems Co., Ltd.). The decomposition product at a first
step was analyzed by a high speed liquid chromatography,
where 200 picomoles of PTH-alanine was detected but other
PTH-amino acids were not detected. Thus, the N-terminal
amino acid of IL-2 was identified to be alanine. From the
decomposition product at a second step, 180 picomoles of
PTH-proline and a small quantity of PTH-alanine were
detected but no other PTH amino acids were detected; thus,
a second amino acid from the N-terminal of IL-2 was
identified as proline. From the decomposition product at
a third step, 30 picomoles of PTH-threonine and a small
quantity of PTH-proline were detected but no other
PTH-amino acids were detected; thus a third amino acid from
the N-terminal of IL-2 was identified as threonine. It
is known that PTH-threonine is unstable and liable to be
decomposed; a low recovery rate of PTH-threonine is often
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experienced in the art. From the decomposition products
from 4th, 5th, 6th and 7th steps, 20 to 40 picomoles of
PTH-serine, PTH-serine, PTH-serine and PTH-threonine were
exclusively detected, respectively. It is known that
PTH-serine is also unstable and liable to be decomposed
and for this reason, the recovery rate was poor but no other
PTH-amino acids were detected; thus, the 4th to 7th amino
acids from the N-terminal of IL-2 were identified as serine,
serine, serine and threonine, respectively. From the decom-
position products at 8th, 9th and 10th steps, 100 picomoles
of PTH-lysine, 120 picomoles of PTH-lysine and 20 picomoles
of PTH-threonine were detected, respectively and the 8th,
9th and 10th amino acids from the N-terminal of IL-2 were
identified as lysine, lysine and threonine, respectively.
In a similar fashion, the 11th to 15th amino acids from
the N-terminal of IL-2 were identified as glutamine, leucine,
/acid
glutamine, leucine and glutamic respectively, whereby the
detected data of the corresponding PTH amino acids were
60 to 120 picomoles.
From the decomposition product at a 16th step,
20 picomoles of PTH-histidine were detected. It is known
that PTH-histidine is also poor in the recovery rate. In
a similar fashion, from the decomposition products at 17th
to 30th from the N-terminal of IL-2 were identified as
leucine, leucine, leucine, aspartic acid, leucine,
glutamine, methionine, isoleucine, leucine, asparagine,
glycine, isoleucine, asparagine and
asparagine, respectively. The partial amino acid sequence
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of IL-2 is fully identical with that anticipated by the
base sequence of gene.
Next, the C-terminal amino acid of IL-2 obtained
was determined. The determination of the C-terminal was
performed in a manner similar to the method of Chang et al
using carboxypeptidase Y (Biochem. J., 199, 547-555 (1981)).
In 30 p.2 of a 0.05 M acetic acid buffer solution (pH 5.4)
was dissolved about 80 pg (500 picomoles) of IL-2 and 1 p
of a 0.1 mg/ml solution of carboxypeptidase Y was added to
the solution and the resulting mixture was maintained at
25 C. From the reaction liquid, 7 pi each of samples was
taken with the lapse of time. After freeze drying each
of the samples, 10 pi of a 0.1 M NaHCO3 (adjusted pH to
9.0) was added thereto. Next, 20 p.2 of a 4 mmole/.¾
acetone solution of dimethylaminoazobenzenesulfonyl chloride
purified from recrystallization was added. After heating
the mixture at 70 C for 15 minutes, 200 pi of 70/10' ethanol
was added. Using 10 pf out of the mixture, HPLC analysis
was performed. As a result of the HPLC analysis,
dimethylaminoazobenzenesulfonyl (hereafter simply referred
to as DABS)-threonine was detected from the reaction liquid
at the initial stage and DABS-leucine was detected somewhat
later; thus, the C-terminal amino acid of IL-2 was identified
as threonine and the amino acid sequence around the C-
terminal as leucine-threonine (C-terminal).
From the above experimental results, it was found
that the amino acid sequences around the N-terminal and
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X341633
C-terminal of IL-2 obtained were completely identical with
those anticipated from the base sequence of a gene and the
compositional ratio of the constituent amino acids was
then examined.
In a conventional manner about 40 pg (250 pico-
moles) of IL-2 was hydrolyzed in 6N HC1 at 110 C for 48
hours and the analysis was conducted using an amino acid
analyzer. The results are shown in the table. With respect
to serine, threonine and tryptophan which are known to
cause decomposition under the hydrolysis conditions described
above, serine and threonine were corrected using the
analytical data of hydrolysis at 110 C for 24 hours, and
tryptophan was separately determined by fluorometry. As
is evident from Table 3, the amino acid composition of IL-2
obtained was identical with that expected from the base
sequence of a gene. From the above results, it is judged
that a primary structure of IL-2 obtained would be as shown
in the amino acid sequence of Formula I.
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Table 3
Compositional Ratio of Amino Acid
Found Value Assumed Value
Aspartic acid 12.5 12
Threonine 12.5 13
Serine 7.7 8
Glutamic acid 18.4 18
Proline 5.2 5
Glycine 2..1 2
Alanine 5.0 5
1/2 Cyst"ine 2.7 3
Valine 4.1 4
Methionine 4.3 4
Isoleucine 8.6 9
Leucine 21.5 22
Tyrosine 2.9 3
Phenylalanine 5.8 6
Lysine 11.3 11
Histidine 3.2 3
Tryptophane 1.0 1
Arqinine 4.1 4
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13 41 633
The structure of IL-2 was further confirmed by measuring
.the molecular weights of two kinds of decomposition products of
IL-2 by means of mass spectrometry. Fifteen micrograms of IL-2
(about 1 nmole) was dissolved in 14 p.1 of 70 % formic acid,
added with 46 p-g of cyanogen bromide, which cleaves a carboxyside
o.f methionine residue and the methionine is converted to homose-
rine or homoserine lactone, in 1 p1 of 70 % formic acid, and then
allowed to stand overnight at a room temperature. The reaction
mixture was evaporates in vacuo, added with 40p.g of water, and
then lyophilized. The product was added with 14,u1of 1 % ammonium
bicarbonate and then digested with 0.3,ijg of trypsin (Warthington
Co.,), to cleave a carboxyside of lysine or arginine residue,in
0.6 ,u1 of 1 % ammonium bicarbonate at 37 C. One third aliquot of
the reaction mixture was withdrawn after 3 hr or 6 hr incubation,
added with 0.5 l of acetic acid, and then subjected to mass
spectrometry. Another 15JLg of IL-2 was.also threated by the
same method as mentioned above except digesting with 0.3 #g of
Stdphylococcus aureus D8 protease (Miles Co.,), to cleave a
carboxyside of glutamic acid residue, instead of trypsin.
Molecular weights of the decomposition products in a mixture
-state were measured by means of fast atom bombardment mass
spectrometry on a JMS-HX100 spectrometer (JEOL CO.). Many kinds
of molecular ion peaks accompanied by isotopic peaks were observed
on mass spectra.
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Representataive peaks corresponding to the molecular
weights (MH'-) of cyanogen bromide-trypsin decomposition produces of
IL-2 are shown in Table 4.
Table 4
molecular weight identification
m/z 1783 Lys-9 to Met-23
m/z 1665 Thr-10 to Met-23
m/z 1049 Ile-24 to Lys-32
m/z 389 Leu-36 to Arg-38
m/z 508 Leu-40 to Lys-43
m/z 561 Ala-50 to Lys-54
m/z 2564 His-55 to Lys-76
m/z 939 AsN-77 to Arg-83
m/z 1583 Asp-84 to Lys-97
m/z 1874 Cys-105 to Arg-120
Representative peaks corresponding to the molecular
weights (MH+) of cyanogen bromide-V8 protease decomposition
)roducts of IL-2 are shown in Table 5.
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13 41 633
Table 5
molecular weight identification
m/z 1619 Ala-1 to Glu-15
m/z 952 His-16 to Met-23
m/z 1859 Ile-24 to Met-39
m/z 919 Leu-40 to Met-46
m/z 1241 Leu-53 to Glu-62
m/z 857 Glu-61 to Glu-67 or Glu-62 to Glu-68
m/z 728 Glu-62 to Glu-67 or Glu-63 to Glu-68
m/z 3116 Val-69 to Glu-95
m/z 633 Thr-111 to Glu-116
Above data show that 83 % of the primary structure of
IL-2 was identified (Ala-1 to Met-46, Ala-50 to Lys-97 and
Cys-105 to Arg-120; i.e., 110 amino acid residues in 133 amino
acid residues of IL-2) by mass spectrometry, ascertaing the
promary structure of IL-2 of the amino acid sequence of Formula I.
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Example 2
(1) A plasmid which should direct the synthesis
of human IL-2 in E. coli cells was constructed as follows.
A plasmid pTIL2-22 was constructed from pTrS-3 (Nishi T.,
Taniguchi T. et al., SEIKAGAKU 53, 967, (1981)), and pIL
2-50A containing the IL-2 cDNA by a series of modification
procedures as illustrated in Fig. 5. A plasmid pTrS-3
include insertion of the region of T_rp promoter and Shine
Dalgarno (hereinafter "SD") between EcoRI site and Clal
site of pBR 322. The plasmid also contains an ATG initiation
codon 13 bp downstream of the SD sequence as well as a single
SphI site as illustrated in Fig. 3. The vector is very
efficient to produce the said protein when DNA sequence
corresponding to the said protein is inserted in phase just
downstream of the ATG codon, which is generated by SphI
digestion and by subsequent treatment by T4 DNA polymerase
of pTrS-3. Therefore the plasmid pTrS-3 (30 pg) was cleaved
with a restriction enzyme SphI in a conventional manner and
after successive treatment with phenol and chloroform,
ethanol precipitates were recovered, then both ends were
rendered flush by the treatment of T4 DNA polymerase. Then
the DNA (21.4 pg) was recovered by similar successive phenol,
chloroform treatment and ethanol precipitation. On the
other side, 380 pg of pIL 2-50A containing an IL-2 cDNA
was cleaved by PstI and the IL-2 cDNA insert was isolated
by agarose gel electrophoresis. cDNA insert (11 ug) was
cleaved by HgiAI, treated by T4 DNA polymerase and 10 vg
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;341633
of the DNA of larger site was isolated by agarose gel
electrophoresis. According to the procedures a cDNA (7.2
pg) coding for 132 amino acids was obtained and this DNA
fragment had blunt ends (Fig. 5). Then the thus obtained
cDNA fragment ligated to a pTrS-3 vector, previously digested
by SphI and treated by T4 DNA polymerase just downstream
of ATG sequence. Thus ligated plasmid was then used to
transform into E. coli HB101 according to the conventional
procedures. Ligation was carried out as follows. IL-2
cDNA (0.4 pg) larger fragment and 0.2 jig of pTrS-3 vector
DNA were mixed with 0.8 unit of T4 DNA ligase in 66 mM
Tris-HC1 of pH 7.5 containing 6.6 mM MgC12, 1 mM ATP and
mM DTT, and the mixture was allowed to react at 4 C
overnight. Among the transformants appeared on L broth
agar plate containing ampicillin, colonies containing the
IL-2 cDNA portion, which encodes 132 amino acids were
selected by in situ colony hybridization assay. Thus
selected colonies were cultured (10 ml) again to prepare
plasmid DNA by lysozyme treatment and by freeze-thawing.
The plasmid DNAs were cleaved with PstI and Xbal, and the
resulting products were analysed by agarose gel electro-
phoresis in order to identify pTIL 2-22 in which the cDNA
was linked to the ATG sequence of pTrS-3 in correct
orientation. The E. coli HB101 containing pTIL 2-22 was
cultured under the conventional conditions known for the
propagation of microorganisms. The cells were grown in
10 ml of X broth (2.5 6 Bactotrypton, 1.0 6 yeast extracts,
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1341633
0.1% glucose, 20 mM MgSO4, 50 mM Tris-HC1, pH 7.5)
containing 25 pg/mi streptomycin and 25 pg of ampicillin
at 37 C for an overnight. One ml of the culture suspension
was inoculated into the same X broth (100 ml) and cultured
at 37 C. When optical density at 650 mp arrived around
1.5-2.0, 50 pg/ml 3-indole acrylic acid (IAA) was added
to the medium. Three hours after the addition of the
inducer, the cells were collected, washed with 20 mM
Tris-HC1 (pH 7.5, 30 mM NaCl) and resuspended into 8 ml
of the same buffer. Thus produced proteins in bacterial
cells were extracted by sonication (0 C 2 min.) followed
with three successive freeze-thawing. The extracted IL-2
activity ranged from 10,000 to 120,000 units/ml.
E. coli HB101 containing pTIL 2-22 (AJ12009) has
been deposited in the accession number of FERM-BP245.
(2) E. coli AJ12009 was cultured by the manner
shown in step (8) of Example 1. A homogenate of the cell
obtained contained 1 x 105 IL-2 activity, and from 160 ml
of the homogenate by the manner shown in step (8) of Example
1, IL-2 polypeptide was recovered in the recovery yield
of 20%, obtaining IL-2 preparation having about 5 x 107
units per 1 mg of IL-2 protein.
The obtained IL-2 polypeptide preparation showed
a single band at the location of a molecular weight of about
16,000 daltons by SDS-polyacrylamide gel electrophoresis.
(3) Using 10 pg of the obtained IL-2, the amino
acids constituting IL-2 were sequentially determined from
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the N-terminal by the method shown in step (9) of Example
1, and found proline as the first N-terminal amino acid
and threonine as the second N-terminal amino acid.
Third to twentieth amino acids from the N-
terminal praline were also found by the manner shown in
step (9) of Example 1 as Ser, ser, Ser, Thr, Lys, Lys,
Thr, Gln, Leu, Gln, Leu, Glu, His, Leu, Leu, Leu, Asp,
and Leu.
C-terminal amino acid of the IL-2 preparation
obtained was also determined by the manner shown in step
(9) of Example 1, and was threonine.
The structure of IL-2 was further confirmed by
measuring the molecular weights of decomposition products
of IL-2 by means of mass spectrometry according to the
same method as in step (A) of Example 1. Similar results
were obtained except for observation of a peak at M/Z
1548 (corresponding to Pro-1 to Glu-14) instead of
M/Z 1619 on a mass spectrum of cyanogen bromide-V8
protease decomposition product.
From the above results, it is judged that a
primary structure of IL-2 obtained would be as shown in
the amino acid sequence of Formula II.
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