AND BIOPHYSICS
`ARCHIVES OF BIOCHEMISTRY
`Vol. 257, No. 1, August
`15, pp. 194-199,1987
`
`Linkage
`of Disulfide
`Importance
`Biologically
`Active Human
`
`for Constructing
`Interleukin-2
`
`the
`
`TAKAO
`
`YAMADA,’
`KOICHI
`
`AKIRA
`KATO,
`
`KENJI
`FUJISHIMA,
`AND OSAMU NISHIMURA
`
`KAWAHARA,
`
`Biotechnology
`
`Laboratories,
`
`Research
`Central
`, Yodogawa-ku,
`
`LXvision,
`Osaka
`532,
`
`Takeda
`.Japan
`
`Ltd
`
`Chemical
`
`Industries,
`
`Received
`
`February
`
`17, 1987, and
`
`in revised
`
`form
`
`April
`
`27,1987
`
`coli possesses a
`in Escherichia
`produced
`(rIL-2)
`interleukin-2
`human
`Recombinant
`free thiol group at Cys-125 and a disulfide linkage between Cys-58 and Cys-105, as in
`the case for natural human interleukin-2. Treatment of rIL-2 with 200 mM dithiothreitol
`resulted in the cleavage of the Cys-58-Cys-105 disulfide bond. The reduced form of rIL-
`2 thus obtained retained only 10% of the in vitro biological activity of the native form,
`as measured by the ability
`to stimulate
`the growth of an IL-Z-dependent mouse natural
`killer cell line, NKCS. Far-uv circular dichroism studies indicated that the cleavage of
`the disulfide bond results in a decrease of a-helix content. Near-uv circular dichroism
`studies suggested that the native molecule is folded into a rigid tertiary structure, while
`the reduced form showed a spectrum similar to that of rIL-2 denatured in the presence
`of 6 M guanidine . HCl. The once-reduced molecule was readily reoxidized in the presence
`to form
`the native molecule with
`full biological activity. These results
`of IO PM
`cU2+
`strongly demonstrate that the Cys-58-Cys-105 disulfide linkage in the IL-2 molecule is
`essential for constructing a rigid and biologically active form of IL-Z.
`o 1987 Academic
`Press, Inc.
`
`Recent advances in recombinant DNA
`technology have led to the mass production
`of eukaryotic proteins by Eschemkhia
`coli
`cells. However, some of recombinant pro-
`teins produced
`in
`this way have been
`proved to be different
`from natural pro-
`teins. Such recombinant proteins as human
`interferon-a
`(1, Z), human interferon-y
`(3),
`human growth hormone (4), and human
`interleukin-2
`(IL-2)2 (5-8) possess an ad-
`ditional methionine residue at their amino
`termini corresponding to the initiator me-
`thionine codon. In addition, recombinant
`calf prochymosin
`is present as an oligo-
`
`be addressed.
`should
`correspondence
`1 To whom
`interleukin-2;
`rIL-2,
`used:
`IL-2,
`’ Abbreviations
`combinant
`interleukin-2;
`DTT,
`dithiothreitol;
`trifluoroacetic
`acid, DTNB,
`5,5’-dithiobis(2-nitroben-
`zoic acid).
`
`re-
`TFA,
`
`merit form interlinked partly by disulfide
`bonds and as a reduced form in inclusion
`bodies of E. coli cells (9). Harris thinks that
`several normally soluble proteins are in-
`soluble in E. coli cells because they
`lack
`proper disulfide bridges or have incorrect
`disulfide bridges (10). It is well known that
`the formation of proper disulfide bonds is
`often essential to the structure and func-
`tion of proteins. Therefore,
`it is important
`to obtain information on disulfide bridges
`of E. co&derived
`recombinant proteins.
`IL-2 is a lymphokine produced by acti-
`vated T lymphocytes and plays important
`roles in the proliferation and differentia-
`tion of T lymphocytes, as well as in the
`regulation of the immune systems (11-13).
`We have purified and characterized recom-
`binant human IL-2 (rIL-2) produced in E.
`coli cells harboring
`the human IL-2 gene
`(5). More recently, we have succeeded in
`
`$3.00
`0003-9861/87
`Press,
`Copyright
`0 1987 by Academic
`All
`rights
`of reproduction
`in any
`form
`
`Inc.
`resewed.
`
`194
`
`Page 1
`
`KASHIV EXHIBIT 1053
`IPR2019-00797
`
`

`

`IMPORTANCE
`
`OF DISULFIDE
`
`LINKAGE
`
`FOR
`
`ACTIVE
`
`HUMAN
`
`INTERLEUKIN-2
`
`195
`
`IL-2 and
`human
`recombinant
`separating
`the dif-
`its methionylated
`form by utilizing
`(14). We
`ference of their
`isoelectric
`points
`of the
`lo-
`report
`here
`the determination
`in E. coli-
`cation of the disulfide
`linkage
`of the di-
`derived
`rIL-2.
`The
`importance
`a rigid
`form
`sulfide
`linkage
`for constructing
`of the IL-2 molecule with
`full biological ac-
`tivity
`is discussed.
`
`MATERIALS
`
`AND
`
`METHODS
`
`in
`rIL-2 produced
`of rIL-2.
`Puri,fication
`the
`E. coli N4830/pTB
`285 cells harboring
`human
`IL-2 gene was purified
`(5) and sep-
`arated
`from
`the contaminating
`methionyl
`rIL-2,
`as described
`previously
`(14).
`Isolation
`of thiol-containing
`peptide frag-
`ments. The thiol-containing
`peptides were
`isolated, essentially
`in the same manner as
`et al. (15).
`that
`reported
`by Egorov
`rIL-2
`(6.3 mg, 420 nmol) dissolved
`in 3 ml of 6 M
`guanidine. HCl-0.1
`M NaCl-25
`mM am-
`monium acetate
`(pH 5.0) was applied at a
`flow
`rate of 5 ml/h
`to a Thiopropyl-Seph-
`arose 6B (Pharmacia)
`column
`(1.0 X 5.1 cm)
`equilibrated
`with
`the same buffer. The col-
`umn was
`successively
`washed
`with
`the
`equilibration
`buffer,
`0.1 M NaCl-25 mM
`ammonium
`acetate
`(pH
`5.0) and 0.2 M
`acetic acid
`(pH 3.0). The gel was
`trans-
`ferred
`to a tube with
`6 ml of 0.2 M acetic
`acid (pH 3.0) containing
`0.1 mg of pepsin
`(EC 3.4.23.1; Sigma), and then the tube was
`rotated end over end for 15 h at 3’7°C. After
`peptic digestion,
`the gel was again packed
`in a column. The column was washed with
`0.2 M acetic acid (pH 3.0) and 25 mM am-
`monium acetate
`(pH 4.5), and then
`the re-
`sidual 2-thiopyridyl
`groups of the gel were
`removed as 2-thiopyridone
`with 20 mM
`2-
`mercaptoethanol-25
`ammonium
`ace-
`mM
`tate (pH 4.5). The thiol-containing
`peptides
`were eluted at a flow
`rate of 5 ml/h with
`20 mM 2-mercaptoethanol-25
`ammo-
`mM
`nium acetate
`(pH 8.0) after
`the column was
`washed with 25 mM ammonium
`acetate (pH
`4.5) and 25 mM ammonium
`acetate
`(pH 8.0).
`The eluate was
`taken
`to dryness
`with
`a
`Speed Vat
`concentrator
`(Savant
`Instru-
`ments, U.S.A.). The powder
`thus obtained
`was dissolved
`in 0.1% TFA and subjected
`to reverse-phase
`HPLC with
`a Nucleosil
`
`(0.8 X 30 cm, Macherey-Nagel
`5C18 column
`Company,
`FRG). As a result,
`137 nmol of
`four
`thiol-containing
`peptides
`(13 nmol of
`P-l, 48 nmol of P-2,14 nmol of P-3, and 62
`nmol of P-4) was
`isolated starting
`from 420
`nmol of rIL-2, as estimated
`on the basis of
`amino acid analysis.
`rIL-
`of r&2.
`Reduction
`and reoxidation
`solu-
`a rIL-2
`2 was
`reduced by incubating
`for 12
`tion
`in the presence of 200 mM DTT
`The
`h at pH 5.0 and at room
`temperature.
`reaction mixture was applied
`to a Sephadex
`G-25
`(Pharmacia)
`column
`(2.4 X 20 cm)
`equilibrated
`with
`2 mM DTT-5
`mM
`am-
`monium acetate
`(pH 5.0), and reduced
`rIL-
`2 was collected. Reoxidation
`of reduced
`rIL-
`2 was performed
`as follows. Reduced
`rIL-
`2 obtained above was applied
`to a Sephadex
`G-25 column
`(2.4 X 20 cm) equilibrated
`with 10 pM CuS04-5 mM ammonium
`acetate
`(pH 5.0). The eluate was kept at 4°C for 20
`h and
`then
`subjected
`to
`reverse-phase
`HPLC with an Ultrapore
`RPSC column
`(1.0
`X 25 cm, Beckman
`Instruments)
`(5). The
`reoxidized
`form
`thus obtained was
`lyoph-
`ilized and dissolved
`in 5 mM ammonium
`acetate
`(pH 5.0). The protein
`recovery was
`about 70%.
`of the free
`Quantitative
`thiol
`analysis
`group. The free thiol group was determined
`by the method of Ellman
`(16) with
`cysteine
`as a standard. Reduced
`rIL-2 was subjected
`to analysis
`after DTT
`in its preparation
`was
`removed by reverse-phase
`HPLC.
`Assay of IL-2 activity.
`was
`IL-2-activity
`the
`determined
`by the ability
`to stimulate
`cell
`growth
`of an IL-2-dependent
`murine
`line, NKCS, as reported
`previously
`(1’7).
`Assay of protein. Protein was determined
`spectrophotometrically
`in 5 mM
`ammo-
`nium acetate
`(pH 5.0) based on the molar
`absorption
`coefficient
`of rIL-2
`at 280 nm,
`9.58 X
`lo3 M-l
`cm-’
`(5). Protein
`in the prep-
`aration
`of reduced
`rIL-2 was determined
`by the method of Bradford
`(18) using Bio-
`Rad protein assay
`reagent with
`rIL-2 as a
`standard.
`The protein of the standard was
`determined
`by absorbance
`at 280 nm as de-
`scribed above.
`of amino acid composition.
`The
`Analysis
`on
`amino acid composition was determined
`a 24-h hydrolysate
`with
`6 N HCI at 110°C
`in
`the presence
`of 4%
`thioglycolic
`acid.
`
`Page 2
`
`

`

`196
`
`YAMADA
`
`ET AL.
`
`0.05OC
`
`-
`
`was
`using ninhydrin
`Amino acid analysis
`performed on a Hitachi Model 835 amino
`acid analyzer. Cys was determined as cys-
`-
`teic acid on a 24-h hydrolysate after per-
`z
`formic acid oxidation.
`i ,,. ,,25 _
`(CD) studies. CD
`Circular dichroism
`spectra were determined at room temper- G
`ature on a Jasco Model J-20A spectropo-
`g
`larimeter. Spectral bandwidth was set at
`i
`yI
`1 nm. The light path for 200 to 250 nm was
`0.022 cm and that
`for 250 to 310 nm was 9 o
`1.0 cm. The solvent spectrum was obtained
`and subtracted
`from the protein spectrum
`for each sample. The data were expressed
`as the mean residue ellipticity
`(fl), which
`was calculated using
`the mean residue
`weight of IL-2 of 116.
`
`; n jt -
`
`60
`
`nradon
`
`Time
`
`100
`
`80
`(min)
`
`FIG. 1. Reverse-phase
`thiol-containing
`of
`HPLC
`was performed
`rIL-2.
`Elution
`peptides
`derived
`from
`concentration
`of acetonitrile
`with
`a linear
`gradient
`rate was 3.0 ml/
`in
`the presence
`of 0.1% TFA.
`Flow
`min. Peaks, P-l, P-2, P-3, and P-4, were
`subjected
`to
`amino
`acid
`analysis
`and
`indentified
`as
`follows.
`P-l
`(Cys-125~Ser-130);
`Ser
`(1.5), Glx
`Cys
`(0.7),
`Ile
`(l.O),
`(1.5). P-2
`(Cys-125-Thr-131);
`Thr
`(0.9), Ser
`(1.6), Glx
`(lo),
`Cys (0.8), Be (1.5). P-3
`(Cys-125-Ile-129);
`Ser (0.9),
`Glx
`(l.O),
`Cys
`(0.8), Be
`(1.5). P-4
`(Cys-125-Thr-133);
`Thr
`(1.7), Ser
`(1.6), Glx
`(l.O),
`Cys
`(0.8),
`Ile
`(1.5),
`(0.9).
`
`Leu
`
`column to give four peaks, P-l, P-2, P-3,
`and P-4 (Fig. 1). Based on amino acid anal-
`ysis, P-l, P-2, P-3, and P-4 were identified
`as Cys-125-Ser-130, Cys-125-Thr-131, Cys-
`125-Ile-129, and Cys-125-Thr-133,
`respec-
`tively, all of which contained Cys-125 (Fig.
`2). The results demonstrate that the rIL-2
`molecule possesses a free thiol group at
`Cys-125 and a disulfide
`linkage between
`Cys-58 and Cys-105, as in the case for nat-
`ural human IL-2 (19).
`Treatment of rIL-2 with 200 mM DTT re-
`sulted in the cleavage of the Cys-58-Cys-
`105 disulfide linkage in the rIL-2 molecule.
`The reduced form of rIL-2
`thus obtained
`retained only about 10% of the biological
`
`130
`125
`-Cys-Cln-SW-lie-lie-Ser-Thr-Leu-Thr-OH
`
`133
`
`--P-1-
`
`a
`
`-p-3+
`
`PP-4
`
`p-2-
`
`4
`
`RESULTS
`
`Location of Disuljide Linkage
`
`The rIL-2 molecule is composed of 133
`amino acids and contains the three cysteine
`residues, Cys-58, Cys-105, and Cys-125 (14).
`The quantitative analysis of the free thiol
`group by the DTNB method (16) showed
`that one cysteine residue possesses a free
`thiol group (see Table I), while the other
`two cysteine residues are linked together
`via a disulfide bridge. To determine the lo-
`cation of the disulfide linkage, rIL-2 was
`applied on Thiopropyl-Sepharose
`and gel-
`bound rIL-2 was digested by pepsin. The
`thiol-containing
`peptide fragments were
`eluted from
`the gel and subjected to re-
`verse-phase HPLC with a Nucleosil 5C18
`
`TABLE
`
`I
`
`BIOLOGICAL
`NUMBEROFFREETHIOLGROUPSAND
`ACTIVITYFORTHREE FORMSOF rIL-2
`
`rIL-2
`form
`
`Native
`Reduced
`Reoxidized
`
`free
`of
`Number
`groups
`thiol
`per molecule
`
`0.91
`2.62
`0.90
`
`Biological
`activity
`(U/w)
`
`34,000
`3100
`35,500
`
`free
`of
`Note. Number
`activity
`were determined
`and Methods.
`
`groups
`thiol
`as described
`
`and biological
`under Materials
`
`FIG. 2. Amino
`region
`in rIL-2.
`in Fig. 1.
`
`of carboxyl-terminal
`sequence
`acid
`Each peptide
`refers
`to the peaks
`
`shown
`
`Page 3
`
`

`

`IMPORTANCE
`
`OF
`
`DISULFIDE
`
`LINKAGE
`
`FOR
`
`ACTIVE
`
`HUMAN
`
`INTERLEUKIN-2
`
`197
`
`I) and
`(Table
`form
`of the native
`activity
`the native molecule
`was eluted
`later
`than
`on reverse-phase
`HPLC
`(Fig. 3). The cell-
`free extract of E coli cells, when subjected
`to reverse-phase HPLC immediately after
`extraction, gave a major peak not with the
`retention
`time
`for
`the Cys-58-Cys-105
`linked form but with
`that for the reduced
`form of rIL-2
`(Fig. 3D). The lyophilized
`preparation
`from this peak was found to
`consist of apparently homogeneous recom-
`binant human
`IL-2 as judged by SDS-
`polyacrylamide
`gel electrophoresis. The
`quantitative analysis of the free thiol group
`in this preparation
`indicated that the peak
`arose from
`reduced rIL-2.
`In addition,
`treatment of the cell-free extract with DTT
`resulted in little change in the peak area.
`These results, therefore, suggest that re-
`combinant human IL-2 is present as a re-
`duced form in E. coli cells..
`
`A
`
`__--
`
`___---
`
`y----’
`
`__iL-
`I3
`
`0.01
`
`0
`
`0.01
`
`A
`
`0.01
`
`C
`
`-0
`I
`-
`
`E
`
`1
`r
`
`\
`
`I
`
`. . . . . . . . . . . . . . . . . . . ..-.
`
`3 0
`z
`.I
`u
`? N
`8
`g-1
`s
`zr
`‘0
`x
`--2
`”
`
`-
`
`-
`
`200
`
`210
`
`220
`Wavelength
`
`230
`
`240
`(nm)
`
`of rIL-
`forms
`of various
`CD spectra
`FIG. 4. Far-uv
`acetate
`(pH
`2. -,
`Native
`form
`in 5 mM ammonium
`in 2 mM DTT/5
`5.0)
`(1.03 mg/ml);
`---,
`reduced
`form
`-,
`mM ammonium
`acetate
`(pH
`5.0)
`(0.945 mg/ml);
`-.
`reoxidized
`form
`in 5 mM ammonium
`acetate
`(pH
`5.0)
`(0.692 mg/ml);
`* * ., denatured
`in 6 M guani-
`dinemHC1/5
`mM ammonium
`(pH
`5.0)
`(0.796
`mg/ml).
`
`form
`acetate
`
`CD Spectrum Analysis
`
`To compare the structure of the reduced
`form with that of native rIL-2, CD studies
`were carried out. Far-uv CD spectra (Fig.
`4) indicated
`that
`the ordered structures
`were present in both forms, which showed
`a minimum at 208 nm and a shoulder
`around 222 nm, as described for native rIL-
`2 previously by Cohen et al. (20). The rIL-
`2 molecule contains 60% a-helix, 36% /3-
`sheet, and 4% remainder as estimated by
`the method of Provencher et al. (21), while
`the reduced form is poorer in the a-helix
`content than is the native form (Fig. 4). In
`near-uv CD spectra, native rIL-2 exhibited
`positive bands at 266 and 291 nm and a
`broad positive band between 270 and 285
`nm (Fig. 5). The results suggest that
`the
`native form is folded into a rigid tertiary
`structure. On the other hand, the spectrum
`of the reduced form was quite similar
`to
`that of rIL-2 denatured in the presence of
`6 M guanidine . HCl (Fig. 5), indicating that
`reduced rIL-2 fails to have a rigid tertiary
`structure. These results reveal
`that
`the
`Cys-58-Cys-105 disulfide
`linkage of IL-2
`plays an indispensable role for creating a
`
`30
`(min)
`
`P
`NO
`z
`0 0.01
`:
`2
`k
`9”
`
`20
`
`Retention
`
`Time
`
`(0 F-y-?-L
`( 1 10
`
`re-
`(A),
`form
`of native
`HPLC
`FIG. 3. Reverse-phase
`and cell-
`(C) of rIL-2,
`form
`duced
`form
`(B),
`reoxidized
`applied
`(D). Samples
`were
`free extract
`of E. coli cells
`to an Ultrapore
`RPSC
`column
`(1.0 X 25 cm, Beckman
`Instruments).
`The amounts
`of protein
`used were
`.Ol
`mg
`(A, B, and C) and
`1.0 mg
`(D). Elution
`was per-
`formed
`with
`a linear
`gradient
`of acetonitrile
`concen-
`tration
`in
`the presence
`of 0.1% TFA.
`Flow
`rate was
`3.0 ml/min.
`
`Page 4
`
`

`

`198
`
`I
`
`260
`
`270
`
`280
`Wavelength
`
`290
`(nm)
`
`300
`
`FIG. 5. Near-w
`2. -,
`Native
`form
`5.0)
`(1.82 mg/ml);
`mM ammonium
`reoxidized
`form
`(0.692 mg/ml);
`dine. HC1/5 mM
`mg/ml).
`
`of rIL-
`forms
`of various
`CD spectra
`acetate
`(pH
`in 5 mM ammonium
`in 2 mM DTT/5
`---,
`reduced
`form
`acetate
`(pH
`5.0)
`(1.89 mg/ml);
`-a -,
`in 5 mM ammonium
`acetate
`(pH 5.0)
`. . *, denatured
`form
`in 6 M guani-
`ammonium
`acetate
`(pH
`5.0)
`(0.796
`
`active
`
`form of the IL-
`
`rigid and biologically
`2 molecule.
`reoxidized
`readily
`form was
`The reduced
`in the presence of 10 PM Cu’+. The reoxi-
`dized
`form
`thus obtained
`possessed
`the
`same specific activity
`as that of the native
`form
`(Table
`I). Its CD spectra
`(Figs. 4 and
`5) and its elution profile on reverse-phase
`HPLC
`(Fig. 3) were exactly
`the same as
`those
`for
`the native
`form. Analysis
`of the
`free
`thiol group
`indicated
`that
`the reoxi-
`dized form contains
`a disulfide
`linkage be-
`tween Cys-58 and Cys-105, as in the case
`for
`the native
`form.
`
`DISCUSSION
`
`in
`linkage
`of the disulfide
`The location
`rIL-2 was determined
`by pepsin digestion
`of Thiopropyl-Sepharose-bound
`rIL-2. This
`method was quite effective
`in that only the
`thiol-containing
`peptide
`fragments
`were
`isolated.
`It should
`be emphasized
`that
`acidic conditions
`throughout
`the chroma-
`tography
`including
`pepsin digestion
`pre-
`vent
`the artifactual
`disulfide exchange
`re-
`action.
`In
`fact, we
`found
`the disulfide
`
`YAMADA
`
`ET AL.
`
`I
`
`in the chro-
`occurred
`reaction
`exchange
`under neutral or basic condi-
`matography
`tions. The experimental
`results
`indicated
`that
`the rIL-2 molecule contains a free thiol
`group at Cys-125 and a disulfide bridge be-
`tween Cys-58 and Cys-105,
`as is the case
`for natural
`human
`IL-2 produced
`by
`the
`JURKAT
`cell line (19). Peptide mapping of
`rIL-2 with pepsin confirmed
`this conclusion
`(data not shown).
`Robb et al. reported that treating natural
`human IL-2 with DTT resulted in a 70%
`loss of its biological activity
`(19). It
`is
`known that the mutant rIL-2 proteins with
`Cys-58 or Cys-105 substituted by other
`amino acid residues have little biological
`activity
`(22,23). We found that the reduced
`form of rIL-2 retained only 10% of the bio-
`logical activity of the native form. The ac-
`tual biological activity of the reduced form
`might be lower if reoxidation
`into the na-
`tive form during the bioassay is taken into
`consideration. The reduced form was eluted
`as a more hydrophobic derivative
`than the
`native
`form on reverse-phase HPLC. Re-
`verse-phase HPLC analysis indicated that
`recombinant human IL-2 is present as a
`reduced form in E. coli cells. It might be
`kept under inadequately oxidative condi-
`tions in inclusion bodies (24) because of its
`high level of expression. Some of this re-
`duced form
`is gradually activated by the
`air oxidation during the purification pro-
`cess to form the Cys-5%Cys-105 linkage.3
`In this study, however, we showed that the
`reduced form can be rapidly oxidized in the
`presence of 10 PM Cu2+ to become a native
`form with
`full biological activity.
`This
`method would be very helpful in obtaining
`a good yield of Cys-5%Cys-105-linked
`rIL-
`2 from the cell-free extract of E. coli cells.
`Far-uv CD studies suggested that
`the
`ordered structures are present in both na-
`tive and reduced forms and that
`the re-
`duced form has less a-helix content
`than
`the native form. In near-uv CD spectra, the
`native form of rIL-2 showed some CD sig-
`nals derived
`from asymmetric environ-
`ments
`for
`the aromatic
`residues (25),
`
`’ T. Yamada
`
`and K. Kato,
`
`unpublished
`
`results.
`
`Page 5
`
`

`

`IMPORTANCE
`
`OF DISULFIDE
`
`LINKAGE
`
`FOR
`
`ACTIVE
`
`HUMAN
`
`INTERLEUKIN-2
`
`199
`
`form has
`the native
`that
`indicating
`clearly
`the aro-
`into which
`a tertiary
`structure
`On
`the
`incorporated.
`matic
`residues
`are
`showed
`a
`other hand,
`the
`reduced
`form
`spectrum
`similar
`to that of rIL-2 denatured
`in the presence of 6 M guanidine
`. HCl. This
`evidence
`suggests
`that
`the aromatic
`resi-
`dues are not
`incorporated
`into a rigid
`structure
`and are exposed
`to the solvent
`in
`the reduced
`form. These results,
`therefore,
`strongly
`demonstrate
`that
`the Cys-5%Cys-
`105 disulfide
`linkage
`in the
`IL-2 molecule
`plays an essential
`role
`in constructing
`a
`rigid and biologically
`active
`form of IL-2.
`
`ACKNOWLEDGMENTS
`
`Kaki-
`and Dr. Atsushi
`Sugino
`Dr. Yukio
`thank
`We
`Division
`for
`their
`en-
`Research
`of
`the Central
`numa
`couragement
`and discussion
`throughout
`this work. We
`are also grateful
`to Dr. Kyozo
`Tsukamoto
`and Dr.
`Osamu
`Shiho
`for
`determining
`IL-2
`activity
`and Dr.
`James R. Miller
`for
`reading
`the manuscript.
`
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